Methods and materials for making and using transgenic dicamba-degrading organisms

ABSTRACT

The invention provides isolated and at least partially-purified dicamba-degrading enzymes, isolated DNA molecules coding for dicamba-degrading enzymes, DNA constructs coding for dicamba-degrading enzymes, transgenic host cells comprising DNA coding for dicamba-degrading enzymes, and transgenic plants and plant parts comprising one or more cells comprising DNA coding for dicamba-degrading enzymes. Expression of the dicamba-degrading enzymes results in the production of dicamba-degrading organisms, including dicamba-tolerant plants. Finally, the invention provides a method of selecting transformed plants and plant cells based on dicamba tolerance and a method of selecting or screening transformed host cells, intact organisms and parts of organisms based on the fluorescence of 3,6-dichlorosalicylic acid produced as a result of dicamba degradation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/875,747, filed Sep. 3, 2010, now U.S. Pat. No. 8,119,380 which is adivisional of U.S. application Ser. No. 10/330,662, filed Dec. 27, 2002,now U.S. Pat. No. 7,812,224 which is a divisional of U.S. applicationSer. No. 09/797,238, filed Feb. 28, 2001, now U.S. Pat. No. 7,105,724which is a continuation-in-part of U.S. application Ser. No. 09/055,145,filed Apr. 3, 1998, now U.S. Pat. No. 7,022,896 which claims benefit ofpriority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser.Nos. 60/042,666 and 60/042,941, both filed Apr. 4, 1997. The completedisclosures of all of the above-identified applications are incorporatedherein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “10395-1-4_ST25.txt” having a size in bytes of 50 kb,and created on Jan. 11, 2012. The information contained in thiselectronic file is hereby incorporated by reference in its entiretypursuant to 37 CFR §1.52(e)(5).

FIELD OF THE INVENTION

The present invention relates to transgenic organisms that are able todegrade the herbicide dicamba, including transgenic plants that havebeen made tolerant to dicamba. The invention also relates todicamba-degrading enzymes and to DNA molecules and DNA constructs codingfor dicamba-degrading enzymes. The invention further relates to a methodof controlling weeds in fields of dicamba-tolerant transgenic plants andto a method of removing dicamba from materials contaminated with it(bioremediation). Finally, the invention relates to methods of selectingtransformants based on dicamba tolerance or on detecting thefluorescence of 3,6-dichlorosalicylic acid which is generated as aresult of dicamba degradation.

BACKGROUND

Herbicides are used routinely in agricultural production. Theireffectiveness is often determined by their ability to kill weed growthin crop fields and the tolerance of the cash crop to the herbicide. Ifthe cash crop is not tolerant to the herbicide, the herbicide willeither diminish the productivity of the cash crop or kill it altogether.Conversely, if the herbicide is not strong enough, it may allow too muchweed growth in the crop field which will, in turn, lessen theproductivity of the cash crop. Therefore, it is desirable to produceeconomically important plants which are tolerant to herbicides. Toprotect the water and environmental quality of agricultural lands, it isalso desirable to develop technologies to degrade herbicides in cases ofaccidental spills of the herbicide or in cases of unacceptably highlevels of soil or water contamination.

Genes encoding enzymes which inactivate herbicides and other xenophobiccompounds have previously been isolated from a variety of procaryoticand eucaryotic organisms. In some cases, these genes have beengenetically engineered for successful expression in plants. Through thisapproach, plants have been developed which are tolerant to theherbicides 2,4-dichlorophenoxyacetic acid (Streber and Willmitzer (1989)Bio/Technology 7:811-816; 2,4-D), bromoxynil (Stalker et al. (1988)Science 242:419-423; tradename Buctril), glyphosate (Comai et al. (1985)Nature 317:741-744; tradename Round-Up) and phosphinothricin (De Blocket al. (1987) EMBO J. 6:2513-2518; tradename Basta).

Dicamba (tradename Banvel) is used as a pre-emergent and post-emergentherbicide for the control of annual and perennial broadleaf weeds andseveral grassy weeds in corn, sorghum, small grains, pasture, hay,rangeland, sugarcane, asparagus, turf and grass seed crops. See CropProtection Reference, pages 1803-1821 (Chemical & Pharmaceutical Press,Inc., New York, N.Y., 11th ed., 1995). Unfortunately, dicamba can injuremany commercial crops (including beans, soybeans, cotton, peas,potatoes, sunflowers, tomatoes, tobacco, and fruit trees), ornamentalplants and trees, and other broadleaf plants when it comes into contactwith them. Id. Dicamba is chemically stable and can sometimes bepersistent in the environment.

Dicamba is in the class of benzoic acid herbicides. It has beensuggested that plants tolerant to benzoic acid herbicides, includingdicamba, can be produced by incorporating a1-aminocyclopropane-1-carboxylic acid (ACC) synthase antisense gene, anACC oxidase antisense gene, an ACC deaminase gene, or combinationsthereof, into the plants. See Canadian Patent Application 2,165,036(published Jun. 16, 1996). However, no experimental data are presentedin this application which demonstrate such tolerance.

Bacteria that metabolize dicamba are known. See U.S. Pat. No. 5,445,962;Krueger et al., J. Agric. Food Chem., 37, 534-538 (1989); Cork andKrueger, Adv. Appl. Microbiol., 38, 1-66 (1991); Cork and Khalil, Adv.Appl. Microbiol., 40, 289-320 (1995). It has been suggested that thespecific genes responsible for dicamba metabolism by these bacteriacould be isolated and used to produce dicamba-resistant plants and otherorganisms. See id. and Yang et al., Anal. Biochem., 219:37-42 (1994).However, prior to the present invention, no such genes had beenidentified or isolated.

SUMMARY OF THE INVENTION

The invention provides an isolated and at least partially purifieddicamba-degrading O-demethylase, an isolated and at least partiallypurified dicamba-degrading oxygenase, an isolated and at least partiallypurified dicamba-degrading ferredoxin, and an isolated and at leastpartially purified dicamba-degrading reductase, all as defined anddescribed below.

The invention also provides an isolated DNA molecule comprising a DNAsequence coding for a dicamba-degrading oxygenase, an isolated DNAmolecule comprising a DNA sequence coding for a dicamba-degradingferredoxin, and an isolated DNA molecule comprising a DNA sequencecoding for a dicamba-degrading reductase. The invention further providesa DNA construct comprising a DNA sequence coding for a dicamba-degradingoxygenase, a DNA sequence coding for a dicamba-degrading ferredoxin, ora DNA sequence coding for a dicamba-degrading reductase, each DNA codingsequence being operatively linked to expression control sequences. Inaddition, the invention provides a DNA construct comprising a DNAsequence coding for a dicamba-degrading oxygenase, a DNA sequence codingfor a dicamba-degrading ferredoxin, and a DNA sequence coding for adicamba-degrading reductase, each DNA coding sequence being operativelylinked to expression control sequences.

The invention further provides a transgenic host cell comprising DNAcoding for a dicamba-degrading oxygenase, DNA coding for adicamba-degrading ferredoxin, or DNA coding for a dicamba-degradingreductase, each DNA being operatively linked to expression controlsequences. In addition, the invention provides a transgenic host cellcomprising DNA coding for a dicamba-degrading oxygenase and DNA codingfor a dicamba-degrading ferredoxin, DNA coding for a dicamba-degradingreductase, or DNA coding for a dicamba-degrading ferredoxin and DNAcoding for a dicamba-degrading reductase, each DNA being operativelylinked to expression control sequences. The transgenic host cell may bea plant cell or a prokaryotic or eukaryotic microorganism.

The invention also provides a transgenic plant or plant part comprisingone or more cells comprising DNA coding for a dicamba-degradingoxygenase, DNA coding for a dicamba-degrading ferredoxin, or DNA codingfor a dicamba-degrading reductase, each DNA being operatively linked toexpression control sequences. The invention further provides atransgenic plant or plant part comprising one or more cells comprisingDNA coding for a dicamba-degrading oxygenase and DNA coding for adicamba-degrading ferredoxin, DNA coding for a dicamba-degradingreductase, or DNA coding for a dicamba-degrading ferredoxin and DNAcoding for a dicamba-degrading reductase, each DNA being operativelylinked to expression control sequences. The transgenic plant or plantpart is preferably tolerant to dicamba or has had its tolerance todicamba increased as a result of the expression of the dicamba-degradingenzyme(s).

The invention also provides a method of controlling weeds in a fieldcontaining transgenic dicamba-tolerant plants. The method comprisesapplying an amount of dicamba to the field which is effective to controlthe weeds.

The invention further provides methods of decontaminating a materialcontaining dicamba. In one embodiment, the method comprises applying aneffective amount of a transgenic dicamba-degrading microorganism to thematerial. In another embodiment, the method comprises applying aneffective amount of a dicamba-degrading O-demethylase or of acombination of a dicamba-degrading oxygenase, a dicamba-degradingferredoxin and a dicamba-degrading reductase to the material.

The invention also provides a method of selecting transformed plantcells and transformed plants using dicamba tolerance as the selectionmarker. In one embodiment, the method comprises transforming at leastsome of the plant cells in a population of plant cells so that they aretolerant to dicamba and growing the resulting population of plant cellsin a culture medium containing dicamba at a concentration selected sothat transformed plant cells will grow and untransformed plant cellswill not grow. In another embodiment, the method comprising applyingdicamba to a population of plants suspected of comprising plants thathave been transformed so that they are tolerant to dicamba, the dicambabeing applied in an amount selected so that transformed plants willgrow, and the growth of untransformed plants will be inhibited.

Finally, the invention provides a method of selecting, or screening for,transformed host cells, intact organisms, and parts of organisms. Themethod comprises providing a population of host cells, intact organisms,or parts of organisms suspected of comprising host cells, intactorganisms, or parts of organisms that have been transformed so that theyare able to degrade dicamba, contacting the population of host cells,intact organisms, or parts of organisms with dicamba, and ascertainingthe presence or level of fluorescence due to 3,6-dichlorosalicylic acid.The 3,6-dichlorosalicyclic acid is generated in transformed host cells,intact organisms, or parts of organisms as a result of the degradationof dicamba, but will not be generated in untransformed host cells,intact organisms, or parts of organisms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A diagram of the proposed electron transport scheme for dicambaO-demethylase. Electrons from NADH are transferred sequentially fromreductase_(DIC) to ferredoxin_(DIC) and then to oxygenase_(DIC). Thereaction of oxygen with the substrate dicamba to form3,6-dichlorosalicylic acid is catalyzed by oxygenase_(DIC). ox,oxidized; red, reduced.

FIG. 2. Comparison of the derived amino acid sequence of the ferredoxincomponent of dicamba O-demethylase to the amino acid sequences ofmembers of the adrenodoxin family of ferredoxins. In FIG. 2, ferrdi6=ferredoxin component of dicamba O-demethylase from Pseudomonasmaltophilia DI-6 [SEQ ID NO:5]; fer6 rhoca=ferredoxin from Rhodobactercapsulatus [SEQ ID NO:10]; fer caucr=ferredoxin from Caulobactercrescentus [SEQ ID NO:11]; thcc rhocr=ferredoxin from Rhodococcuserythropolis [SEQ ID NO:12]; putx psepu=ferredoxin from Pseudomonasputida [SEQ ID NO:13]; terp psesp=ferredoxin from Pseudomonas sp. [SEQID NO:14]. Also in FIG. 2, the numbers 1-3 designate the three conservedmotifs of the adrenodoxin family of bacterial ferredoxins.

FIG. 3. Comparison of the derived amino acid sequence of the tworeductase components of dicamba O-demethylase to the amino acidsequences of members of the family of FAD-dependent pyridine nucleotidereductases. In FIG. 3, red1 di6=reductase component of dicambaO-demethylase from P. maltophilia DI-6 [SEQ ID NO:7]; red2 di6=reductasecomponent of dicamba O-demethylase from P. maltophilia DI-6 [SEQ IDNO:9]; AJ002606=reductase from Sphingomonas sp. [SEQ ID NO:15]; thcdrhoer=reductase from R. erythropolis [SEQ ID NO:16]; camapsepu=reductase from P. putida [SEQ ID NO:17]; tera pscsp=reductase fromPseudomonas sp. [SEQ ID NO:18]. Also in FIG. 3, the numbers 1-5designate the five conserved motifs of FAD-dependent pyridine nucleotidereductases.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS OF THEINVENTION

Prior studies (Cork and Kreuger, Advan. Appl. Microbiol. 36:1-56 andYang et al. (1994) Anal. Biochem. 219:37-42) have shown that the soilbacterium, Pseudomonas maltophilia, strain DI-6, is capable ofdestroying the herbicidal activity of dicamba through a single stepreaction in which dicamba (3,6-dichloro-2-methoxybenzoic acid) isconverted to 3,6-dichlorosalicylic acid (3,6-DCSA). 3,6-DCSA has noherbicidal activity and has not been shown to have any detrimentaleffects on plants. In addition, 3,6-DCSA is readily degraded by thenormal bacterial flora present in soil.

The experiments described herein confirm the hypothesis of Yang et al.(see id.) that an O-demethylase is responsible for the conversion ofdicamba to 3,6-DCSA by P. maltophilia strain DI-6 and establish that theO-demethylase is a three-component enzyme system consisting of areductase, a ferredoxin, and an oxygenase. See Examples 1 and 3 whichdescribe in detail the isolation, purification and characterization ofthe P. maltophilia O-demethylase and its three components. The reactionscheme for the reaction catalyzed by the three components of dicambaO-demethylase is presented in FIG. 1. As illustrated in FIG. 1,electrons from NADH are shuttled through a short electron chainconsisting of the reductase and ferredoxin to the terminal oxygenasewhich catalyzes the oxidation of dicamba to produce 3,6-DCSA.

In a first embodiment, the invention provides isolated and at leastpartially purified dicamba-degrading enzymes. “Isolated” is used hereinto mean that the enzymes have at least been removed from the cells inwhich they are produced (i.e., they are contained in a cell lysate). “Atleast partially purified” is used herein to mean that they have beenseparated at least partially from the other components of the celllysate. Preferably, the enzymes have been purified sufficiently so thatthe enzyme preparations are at least about 70% homogenous.

In particular, the invention provides an isolated and at least partiallypurified dicamba-degrading O-demethylase. “Dicamba-degradingO-demethylase” is defined herein to mean a combination of adicamba-degrading oxygenase, a dicamba-degrading ferredoxin and adicamba-degrading reductase, all as defined below.

The invention also provides an isolated and at least partially purifieddicamba-degrading oxygenase. “Dicamba-degrading oxygenase” is definedherein to mean the oxygenase purified from P. maltophilia strain DI-6and oxygenases which have an amino acid sequence which is at least about65% homologous, preferably at least about 85% homologous, to that of theP. maltophilia oxygenase and which can participate in the degradation ofdicamba. “Dicamba-degrading oxygenases” include mutant oxygenases havingthe amino acid sequence of the P. maltophilia oxygenase wherein one ormore amino acids have been added to, deleted from, or substituted for,the amino acids of the P. maltophilia oxygenase sequence. Activity ofdicamba-degrading oxygenases can be determined as described in Examples1 and 3.

The invention further provides an isolated and at least partiallypurified dicamba-degrading ferredoxin. “Dicamba-degrading ferredoxin” isdefined herein to mean the ferredoxin purified from P. maltophiliastrain DI-6 and ferredoxins which have an amino acid sequence which isat least about 65% homologous, preferably at least about 85% homologous,to that of the P. maltophilia ferredoxin and which can participate inthe degradation of dicamba. “Dicamba-degrading ferredoxins” includemutant ferredoxins having the amino acid sequence of the P. maltophiliaferredoxin wherein one or more amino acids have been added to, deletedfrom, or substituted for, the amino acids of the P. maltophiliaferredoxin sequence. Activity of dicamba-degrading ferredoxins can bedetermined as described in Examples 1 and 3.

Finally, the invention provides an isolated and at least partiallypurified dicamba-degrading reductase. “Dicamba-degrading reductase” isdefined herein to mean the reductases purified from P. maltophiliastrain DI-6 and reductases which have an amino acid sequence which is atleast about 65% homologous, preferably at least about 85% homologous, tothat of one of the P. maltophilia reductases and which can participatein the degradation of dicamba. “Dicamba-degrading reductases” includemutant reductases having the amino acid sequence of one of the P.maltophilia reductases wherein one or more amino acids have been addedto, deleted from, or substituted for, the amino acids of the P.maltophilia reductase sequence. Activity of dicamba-degrading reductasescan be determined as described in Examples 1 and 3.

Methods of determining the degree of homology of amino acid sequencesare well known in the art. For instance, the FASTA program of theGenetics Computing Group (GCG) software package (University ofWisconsin, Madison, Wis.) can be used to compare sequences in variousprotein databases such as the Swiss Protein Database.

The dicamba-degrading enzymes of the invention can be isolated andpurified as described in Examples 1 and 3 from P. maltophilia or otherorganisms. Suitable other organisms include bacteria other than P.maltophilia strain DI-6 that degrade dicamba. Several strains of suchbacteria are known. See U.S. Pat. No. 5,445,962; Krueger et al., J.Agric. Food Chem., 37, 534-538 (1989); Cork and Krueger, Adv. Appl.Microbiol., 38, 1-66 (1991); Cork and Khalil, Adv. Appl. Microbiol., 40,289-320 (1995). Other dicamba-degrading bacterial strains can beisolated as were these strains by methods well known in the art.

Preferably, however, the dicamba-degrading enzymes of the invention areprepared using recombinant DNA techniques (see below). In particular,mutant enzymes having the amino acid sequence of the P. maltophiliaenzyme wherein one or more amino acids have been added to, deleted from,or substituted for, the amino acids of the P. maltophilia sequence areprepared in this manner using, for example, oligonucleotide-directedmutagenesis, linker-scanning mutagenesis, mutagenesis using thepolymerase chain reaction, and the like. See Ausubel et al. (eds.),Current Protocols In Molecular Biology (Wiley Interscience 1990) andMcPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press1991).

In a second embodiment, the invention provides isolated DNA moleculescoding for dicamba-degrading enzymes of the invention. “Isolated” isused herein to mean that the DNA molecule has been removed from itsnatural environment or is not a naturally-occurring DNA molecule.Methods of preparing these DNA molecules are well known in the art. See,e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y. (1982), Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y. (1989).

For instance, the DNA molecules of the invention may be isolated cDNA orgenomic clones. The identification and isolation of clones coding forthe dicamba-degrading enzymes of P. maltophilia strain DI-6 aredescribed in Examples 2 and 4-5. Additional clones coding fordicamba-degrading enzymes can be obtained in a similar manner. Theisolated clones, or portions of them, can be used as probes to identifyand isolate additional clones from organisms other than the ones fromwhich the clones were originally isolated. Suitable organisms includebacteria that degrade dicamba. As noted above, in addition to P.maltophilia strain DI-6, several strains of bacteria are known thatdegrade dicamba. See U.S. Pat. No. 5,445,962; Krueger et al., J. Agric.Food Chem., 37, 534-538 (1989); Cork and Krueger, Adv. Appl. Microbiol.,38, 1-66 (1991); Cork and Khalil, Adv. Appl. Microbiol., 40, 289-320(1995).

The DNA molecules of the invention can also be chemically synthesizedusing the sequences of isolated clones. Such techniques are well knownin the art. For instance, DNA sequences may be synthesized byphosphoamidite chemistry in an automated DNA synthesizer. Chemicalsynthesis has a number of advantages. In particular, chemical synthesisis desirable because codons preferred by the host in which the DNAsequence will be expressed may be used to optimize expression. Not allof the codons need to be altered to obtain improved expression, butpreferably at least the codons rarely used in the host are changed tohost-preferred codons. High levels of expression can be obtained bychanging greater than about 50%, most preferably at least about 80%, ofthe codons to host-preferred codons. The codon preferences of many hostcells are known. See, e.g., Maximizing Gene Expression, pages 225-85(Reznikoff & Gold, eds., 1986), PCT WO 97/31115, PCT WO 97/11086, EP646643, EP 553494, and U.S. Pat. Nos. 5,689,052, 5,567,862, 5,567,600,5,552,299 and 5,017,692. The codon preferences of other host cells canbe deduced by methods known in the art. Also, using chemical synthesis,the sequence of the DNA molecule or its encoded protein can be readilychanged to, e.g., optimize expression (e.g., eliminate mRNA secondarystructures that interfere with transcription or translation), add uniquerestriction sites at convenient points, delete protease cleavage sites,etc.

In a third embodiment, the present invention provides DNA constructscomprising DNA coding for a dicamba-degrading enzyme operatively linkedto expression control sequences or a plurality of DNA coding sequences,each coding for a dicamba-degrading enzyme and each being operativelylinked to expression control sequences. “DNA constructs” are definedherein to be constructed (non-naturally occurring) DNA molecules usefulfor introducing DNA into host cells, and the term includes chimericgenes, expression cassettes, and vectors.

As used herein “operatively linked” refers to the linking of DNAsequences (including the order of the sequences, the orientation of thesequences, and the relative spacing of the various sequences) in such amanner that the encoded proteins are expressed. Methods of operativelylinking expression control sequences to coding sequences are well knownin the art. See, e.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y. (1982), Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1989).

“Expression control sequences” are DNA sequences involved in any way inthe control of transcription or translation in prokaryotes andeukaryotes. Suitable expression control sequences and methods of makingand using them are well known in the art.

The expression control sequences must include a promoter. The promotermay be any DNA sequence which shows transcriptional activity in thechosen host cell or organism. The promoter may be inducible orconstitutive. It may be naturally-occurring, may be composed of portionsof various naturally-occurring promoters, or may be partially or totallysynthetic. Guidance for the design of promoters is provided by studiesof promoter structure, such as that of Harley and Reynolds, NucleicAcids Res., 15, 2343-61 (1987). Also, the location of the promoterrelative to the transcription start may be optimized. See, e.g.,Roberts, et al., Proc. Natl Acad. Sci. USA, 76, 760-4 (1979). Manysuitable promoters for use in prokaryotes and eukaryotes are well knownin the art.

For instance, suitable constitutive promoters for use in plants include:the promoters from plant viruses, such as the 35S promoter fromcauliflower mosaic virus (Odell et al., Nature 313:810-812 (1985),promoters of Chlorella virus methyltransferase genes (U.S. Pat. No.5,563,328), and the full-length transcript promoter from figwort mosaicvirus (U.S. Pat. No. 5,378,619); the promoters from such genes as riceactin (McElroy et al., Plant Cell 2:163-171 (1990)), ubiquitin(Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensenet al., Plant Mol. Biol. 18:675-689 (1992)), pEMU (Last et al., Theor.Appl. Genet. 81:581-588 (1991)), MAS (Velten et al., EMBO J. 3:2723-2730(1984)), maize H3 histone (Lepetit et al., Mol. Gen. Genet. 231:276-285(1992) and Atanassova et al., Plant Journal 2(3):291-300 (1992)),Brassica napus ALS3 (PCT application WO 97/41228); and promoters ofvarious Agrobacterium genes (see U.S. Pat. Nos. 4,771,002, 5,102,796,5,182,200, 5,428,147).

Suitable inducible promoters for use in plants include: the promoterfrom the ACE1 system which responds to copper (Mett et al. PNAS90:4567-4571 (1993)); the promoter of the maize In2 gene which respondsto benzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen.Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics243:32-38 (1994)), and the promoter of the Tet repressor from Tn10 (Gatzet al., Mol. Gen. Genet. 227:229-237 (1991). A particularly preferredinducible promoter for use in plants is one that responds to an inducingagent to which plants do not normally respond. An exemplary induciblepromoter of this type is the inducible promoter from a steroid hormonegene, the transcriptional activity of which is induced by aglucocorticosteroid hormone. Schena et al., Proc. Natl. Acad. Sci. USA88:10421 (1991). Other inducible promoters for use in plants aredescribed in EP 332104, PCT WO 93/21334 and PCT WO 97/06269.

Suitable promoters for use in bacteria include the promoter of theBacillus stearothermophilus maltogenic amylase gene, the Bacilluslicheniformis alpha-amylase gene, the Bacillus amyloliquefaciens BANamylase gene, the Bacillus subtilis alkaline protease gene, the Bacilluspumilus xylosidase gene, the phage lambda P_(R) and P_(L) promoters, andthe Escherichia coli lac, trp and tac promoters. See PCT WO 96/23898 andPCT WO 97/42320.

Suitable promoters for use in yeast host cells include promoters fromyeast glycolytic genes, promoters from alcohol dehydrogenase genes, theTPI1 promoter, and the ADH2-4c promoter. See PCT WO 96/23898.

Suitable promoters for use in filamentous fungi include the ADH3promoter, the tpiA promoter, the promoters of the genes encodingAspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase,Aspergillus niger neutral alpha-amylase, A. niger acid stablealpha-amylase, A. niger or Aspergillus awamori glucoamylase, R. mieheilipase, A. oryzae alkaline protease, A. oryzae triose phosphateisomerase, and Aspergillus nidulans acetamidase. See PCT WO 96/23898.

Suitable promoters for use in mammalian cells are the SV40 promoter,metallothionein gene promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, and adenovirus 2 major late promoter. See PCT WO96/23898 and PCT WO 97/42320.

Suitable promoters for use in insect cells include the polyhedrinpromoter, P10 promoter, the Autographa californica polyhedrosis virusbasic protein promoter, the baculovirus immediate early gene 1 promoterand the baculovirus 39K delayed-early gene promoter. See PCT WO96/23898.

Finally, promoters composed of portions of other promoters and partiallyor totally synthetic promoters can be used. See, e.g., Ni et al., PlantJ., 7:661-676 (1995) and PCT WO 95/14098 describing such promoters foruse in plants.

The promoter may include, or be modified to include, one or moreenhancer elements. Preferably, the promoter will include a plurality ofenhancer elements. Promoters containing enhancer elements provide forhigher levels of transcription as compared to promoters which do notinclude them. Suitable enhancer elements for use in plants include the35S enhancer element from cauliflower mosaic virus (U.S. Pat. Nos.5,106,739 and 5,164,316) and the enhancer element from figwort mosaicvirus (Maiti et al., Transgenic Res., 6, 143-156 (1997)). Other suitableenhancers for use in other cells are known. See PCT WO 96/23898 andEnhancers And Eukaryotic Expression (Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 1983).

For efficient expression, the coding sequences are preferably alsooperatively linked to a 3′ untranslated sequence. The 3′ untranslatedsequence contains transcription and/or translation terminationsequences. The 3′ untranslated regions can be obtained from the flankingregions of genes from bacterial, plant or other eukaryotic cells. Foruse in prokaryotes, the 3′ untranslated region will include atranscription termination sequence. For use in plants and othereukaryotes, the 3′ untranslated region will include a transcriptiontermination sequence and a polyadenylation sequence. Suitable 3′untranslated sequences for use in plants include those of thecauliflower mosaic virus 35S gene, the phaseolin seed storage proteingene, the pea ribulose biphosphate carboxylase small subunit E9 gene,the soybean 7S storage protein genes, the octopine synthase gene, andthe nopaline synthase gene.

In plants and other eukaryotes, a 5′ untranslated sequence is alsoemployed. The 5′ untranslated sequence is the portion of an mRNA whichextends from the 5′ CAP site to the translation initiation codon. Thisregion of the mRNA is necessary for translation initiation in eukaryotesand plays a role in the regulation of gene expression. Suitable 5′untranslated regions for use in plants include those of alfalfa mosaicvirus, cucumber mosaic virus coat protein gene, and tobacco mosaicvirus.

As noted above, the DNA construct may be a vector. The vector maycontain one or more replication systems which allow it to replicate inhost cells. Self-replicating vectors include plasmids, cosmids and viralvectors. Alternatively, the vector may be an integrating vector whichallows the integration into the host cell's chromosome of the sequencescoding for the dicamba-degrading enzymes of the invention. The vectordesirably also has unique restriction sites for the insertion of DNAsequences. If a vector does not have unique restriction sites, it may bemodified to introduce or eliminate restriction sites to make it moresuitable for further manipulations.

The DNA constructs of the invention can be used to transform a varietyof host cells (see below). A genetic marker must be used for selectingtransformed host cells (“a selection marker”). Selection markerstypically allow transformed cells to be recovered by negative selection(i.e., inhibiting growth of cells that do not contain the selectionmarker) or by screening for a product encoded by the selection marker.

The most commonly used selectable marker gene for plant transformationis the neomycin phosphotransferase II (nptII) gene, isolated from Tn5,which, when placed under the control of plant regulatory signals,confers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.USA, 80:4803 (1983). Another commonly used selectable marker gene is thehygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,and the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet. 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol. Biol.7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Commonly used genes for screening presumptively transformed cellsinclude β-glucuronidase (GUS), β-galactosidase, luciferase, andchloramphenicol acetyltransferase. Jefferson, R. A., Plant Mol. Biol.Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989), Koncz et al.,Proc. Natl. Acad. Sci. USA 84:131 (1987), De Block et al., EMBO J.3:1681 (1984), green fluorescent protein (GFP) (Chalfie et al., Science263:802 (1994), Haseloff et al., TIG 11:328-329 (1995) and PCTapplication WO 97/41228). Another approach to the identification ofrelatively rare transformation events has been use of a gene thatencodes a dominant constitutive regulator of the Zea mays anthocyaninpigmentation pathway. Ludwig et al., Science 247:449 (1990).

Suitable selection markers for use in prokaryotes and eukaryotes otherthan plants are also well known. See, e.g., PCT WO 96/23898 and PCT WO97/42320. For instance, resistance to antibiotics (ampicillin,kanamycin, tetracyline, chloramphenicol, neomycin or hygromycin) may beused as the selection marker.

According to another aspect of the present invention, tolerance todicamba can be used as a selection marker for plants and plant cells.“Tolerance” means that transformed plant cells are able to grow (surviveand regenerate into plants) when placed in culture medium containing alevel of dicamba that prevents untransformed cells from doing so.“Tolerance” also means that transformed plants are able to grow afterapplication of an amount of dicamba that inhibits the growth ofuntransformed plants.

Methods of selecting transformed plant cells are well known in the art.Briefly, at least some of the plant cells in a population of plant cells(e.g., an explant or an embryonic suspension culture) are transformedwith a DNA construct or combination of DNA constructs providing fordicamba degradation. The resulting population of plant cells is placedin culture medium containing dicamba at a concentration selected so thattransformed plant cells will grow, whereas untransformed plant cellswill not. Suitable concentrations of dicamba can be determinedempirically as is known in the art.

Methods of selecting transformed plants are also known in the art.Briefly, dicamba is applied to a population of plants suspected ofcomprising a DNA construct or a combination of DNA constructs providingfor dicamba degradation. The amount of dicamba is selected so thattransformed plants will grow, and the growth of untransformed plantswill be inhibited. The level of inhibition must be sufficient so thattransformed and untransformed plants can be readily distinguished (i.e.,inhibition must be statistically significant). Such amounts can bedetermined empirically as is known in the art.

Further, the generation of 3,6-DCSA as a result of the degradation ofdicamba can be used for selection and screening. The generation of3,6-DCSA can be readily ascertained by observing the fluorescence ofthis compound, allowing selection and screening of transformed hostcells, intact organisms, and parts of organisms (e.g., microorganisms,plants, plant parts, and plant cells). In this regard, the inventionallows for selection and screening of transformed host cells, intactorganisms, and parts of organisms in the same manner as for greenfluorescent protein (GFP). See U.S. Pat. Nos. 5,162,227 and 5,491,084and PCT applications WO 96/27675, WO 97/11094, WO 97/41228 and WO97/42320, all of which are incorporated herein by reference. Inparticular, 3,6-DCSA can be detected in transformed host cells, intactorganisms, and parts of organisms using conventional spectrophotometricmethods. For instance, microscopes can be fitted with appropriate filtercombinations for fluorescence excitation and detection. A hand-held lampmay be used for benchtop work or field work (see Example 1).Fluorescence-activated cell sorting can also be employed. 3,6-DCSA isexcited at a wavelength of 312-313 nm, with a maximum emissionwavelength of 424 nm.

“Parts” of organisms include organs, tissues, or any other part. “Plantparts” include seeds, pollen, embryos, flowers, fruits, shoots, leaves,roots, stems, explants, etc.

Selection based on dicamba tolerance or dicamba degradation can be usedin the production of dicamba-tolerant plants or dicamba-degradingmicroorganisms, in which case the use of another selection marker maynot be necessary. Selection based on dicamba tolerance or dicambadegradation can also be used in the production of transgenic cells ororganisms that express other genes of interest. Many such genes areknown and include genes coding for proteins of commercial value andgenes that confer improved agronomic traits on plants (see, e.g., PCT WO97/41228, the complete disclosure of which is incorporated herein byreference).

The DNA constructs of the invention can be used to transform a varietyof host cells, including prokaryotes and eukaryotes. The DNA sequencescoding for the dicamba-degrading enzyme(s) and the selection marker, ifa separate selection marker is used, may be on the same or different DNAconstructs. Preferably, they are arranged on a single DNA construct as atranscription unit so that all of the coding sequences are expressedtogether. Also, the gene(s) of interest and the DNA sequence(s) codingfor the dicamba-degrading enzyme(s), when dicamba-tolerance or dicambadegradation is being used as a selection marker, may be on the same ordifferent DNA constructs. Such constructs are prepared in the samemanner as described above.

Suitable host cells include prokaryotic and eukaryotic microorganisms(e.g., bacteria (including Agrobacterium tumefaciens and Escherichiacoli), yeast (including Saccharomyces cerevisiae) and other fungi(including Aspergillus sp.), plant cells, insect cells, and mammaliancells. Preferably, the host cell is one that does not normally degradedicamba. However, the present invention can also be used to increase thelevel of dicamba degradation in host cells that normally degradedicamba.

Thus, the “transgenic” cells and organisms of the invention includecells and organisms that do not normally degrade dicamba, but which havebeen transformed according to the invention so that they are able todegrade this herbicide. The “transgenic” cells and organisms of theinvention also include cells and organisms that normally degradedicamba, but which have been transformed according to the invention sothat they are able to degrade more of this herbicide or to degrade theherbicide more efficiently.

Methods of transforming prokaryotic and eukaryotic host cells are wellknown in the art. See, e.g., Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y. (1982), Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1989);PCT WO 96/23898 and PCT WO 97/42320.

For instance, numerous methods for plant transformation have beendeveloped, including biological and physical transformation protocols.See, for example, Miki et al., “Procedures for Introducing Foreign DNAinto Plants” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pp. 67-88. In addition, vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pp. 89-119.

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium. See, for example, Horsch et al., Science 227:1229 (1985).A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteriawhich genetically transform plant cells. The Ti and Ri plasmids of A.tumefaciens and A. rhizogenes, respectively, carry genes responsible forgenetic transformation of the plant. See, for example, Kado, C. I.,Crit. Rev. Plant. Sci. 10:1 (1991). Descriptions of Agrobacterium vectorsystems and methods for Agrobacterium-mediated gene transfer areprovided by numerous references, including Gruber et al., supra, Miki etal., supra, Moloney et al., Plant Cell Reports 8:238 (1989), and U.S.Pat. Nos. 4,940,838 and 5,464,763.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles. The expression vector is introduced intoplant tissues with a biolistic device that accelerates themicroprojectiles to speeds sufficient to penetrate plant cell walls andmembranes. Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, J.C., Trends Biotech. 6:299 (1988), Sanford, J. C., Physiol. Plant 79:206(1990), Klein et al., Biotechnology 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. USA 84:3962 (1987). Direct uptake of DNAinto protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994).

Transgenic dicamba-tolerant plants of any type may be produced accordingto the invention. In particular, broadleaf plants (including beans,soybeans, cotton, peas, potatoes, sunflowers, tomatoes, tobacco, fruittrees, and ornamental plants and trees) that are currently known to beinjured by dicamba can be transformed so that they become tolerant tothe herbicide. Other plants (such as corn, sorghum, small grains,sugarcane, asparagus, and grass) which are currently considered tolerantto dicamba can be transformed to increase their tolerance to theherbicide. “Tolerant” means that the transformed plants can grow in thepresence of an amount of dicamba which inhibits the growth ofuntransformed plants

It is anticipated that the dicamba-degrading oxygenases of the inventioncan function with endogenous reductases and ferredoxins found intransgenic host cells and organisms to degrade dicamba. Plantchloroplasts are particularly rich in reductases and ferredoxins.Accordingly, a preferred embodiment for the production of transgenicdicamba-tolerant plants is the use of a sequence coding for a peptidethat will direct the dicamba-degrading oxygenase into chloroplasts (“achloroplast targeting sequence”). DNA coding for the chloroplasttargeting sequence is preferably placed upstream (5′) of the sequencecoding for the dicamba-degrading oxygenase, but may also be placeddownstream (3′) of the coding sequence, or both upstream and downstreamof the coding sequence. Exemplary chloroplast targeting sequencesinclude the maize cab-m7 signal sequence (see Becker et al., Plant Mol.Biol. 20:49 (1992) and PCT WO 97/41228) and the pea glutathionereductase signal sequence (Creissen et al., Plant J. 2:129 (1991) andPCT WO 97/41228). An alternative preferred embodiment is the directtransformation of chloroplasts using a construct comprising a promoterfunctional in chloroplasts to obtain expression of the oxygenase inchloroplasts. See, e.g., PCT application WO 95/24492 and U.S. Pat. No.5,545,818. Of course, if a selected transgenic host cell or organismdoes not produce sufficient endogenous reductase, ferredoxin, or both,the host cell or organism can be transformed so that it produces one orboth of these enzymes as well as the oxygenase.

In yet another embodiment, the invention provides a method ofcontrolling weeds in a field where transgenic dicamba-tolerant plantsare growing. The method comprises applying an effective amount ofdicamba to the field to control the weeds. Methods of applying dicambaand amounts of dicamba effective to control various types of weeds areknown. See Crop Protection Reference, pages 1803-1821 (Chemical &Pharmaceutical Press, Inc., New York, N.Y., 11th ed., 1995).

In another embodiment, the invention provides a method of degradingdicamba present in a material, such as soil, water, or waste products ofa dicamba manufacturing facility. Such degradation can be accomplishedusing the dicamba-degrading enzymes of the invention. The enzymes can bepurified from microorganisms naturally expressing them (see Examples 1and 3) or can be purified from transgenic host cells producing them. Ifthe enzymes are used in such methods, then appropriate cofactors mustalso be provided (see Example 1). Effective amounts can be determinedempirically as is known in the art (see Example 1). Alternatively,transgenic prokaryotic and eukaryotic microorganisms can be used todegrade dicamba in such materials. Transgenic prokaryotic and eukaryoticmicroorganisms can be produced as described above, and effective amountscan be determined empirically as is known in the art.

Dicamba is introduced into the environment in large quantities on acontinuing basis. The elimination of dicamba is dependent in large parton the action of enzyme systems which are found in microorganismsinhabiting the soil and water of the planet. An understanding of theseenzyme systems, including dicamba-degrading O-demethylases and theirthree components, is important in efforts to exploit natural andgenetically modified microbes for bioremediation and the restoration ofcontaminated soil, water and other materials. Thus, thedicamba-degrading enzymes, DNA molecules, DNA constructs, etc., of theinvention can be used as research tools for the study of dicambadegradation and bioremediation.

Finally, the dicamba-degrading enzymes of the invention can be used inan assay for dicamba. A sample suspected of containing dicamba is mixedwith a dicamba-degrading O-demethylase or a combination of adicamba-degrading oxygenase, a dicamba-degrading ferredoxin and adicamba-degrading reductase. Suitable assays are described in Examples 1and 3. In particular, detecting or quantitating the fluorescence due tothe generation of 3,6-DCSA makes for a convenient assay.

EXAMPLES Example 1 Purification and Characterization of the Componentsof Dicamba O-Demethylase of Pseudomonas maltophilia DI-6

Methods and Materials:

Bacterium and Growth Conditions.

Pseudomonas maltophilia, strain DI-6 (Kreuger, et al., (1989) J. Agric.Food Chem., 37:534-538) was isolated from a soil site persistentlycontaminated with dicamba. The bacterium was provided by Dr. DouglasCork of the Illinois Institute of Technology (Chicago, Ill.), and wasmaintained on reduced chloride medium (Kreuger, J. P., (1989) Ph.D.thesis, Illinois Institute of Technology, Chicago, Ill.), supplementedwith either dicamba (2 mg/ml) or a mixture of glucose (2 mg/ml) andCasamino Acids (2 mg/ml). The carbon sources were filter-sterilized andadded to the medium after it was autoclaved. Solid media were preparedby the addition of 1% (wt/vol) Gelrite (Scott Laboratories, WestWarwick, R.I.).

Chemicals and Reagents.

Dicamba, 3,6-DCSA, and [¹⁴C]dicamba (U-phenyl-¹⁴C, 42.4 mCi/mmol,radiochemical purity greater than 98%) were supplied by Sandoz Agro,Inc. (Des Plaines, Ill.). To increase solubility, the dicamba and3,6-DCSA stock solutions were prepared by titration with KOH to pH 7.0.All chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.),unless otherwise stated. Superose 12, Mono Q, Q-Sepharose (Fast Flow)and Phenyl-Sepharose (CL-4B) column packings for the FPLC (fastperformance liquid chromatography) apparatus were obtained fromPharmacia (Milwaukee, Wis.). Ampholyte pH 4-6 and ampholyte pH 4-9 werepurchased from Serva (Heidelberg, FRG). Acrylamide, β-mercaptoethanol,N,N,N′,N′-tetramethylethylenediamine (TEMED) and ammonium persulfate(APS) were from Bio-Rad Laboratories (Hercules, Calif.). Thin layerchromatography (TLC) plates were silica gel (250 μm thickness) with UV254 indicator, and were purchased from J. T. Baker Chemical Co.(Phillipsburg, N.J.).

Enzyme Assays.

Dicamba O-demethylase activity was assayed by measuring the formation of[¹⁴C]3,6-DCSA from [¹⁴C]dicamba. Briefly, the activity in mixtures ofenzyme components was measured at 30° C. in a standard 300 μl reactionmixture containing 25 mM potassium phosphate buffer (pH 7.0), 10 mMMgCl₂, 0.5 mM NADH (beta-nicotinamide adenine dinucleotide, reducedform), 0.5 mM ferrous sulfate, 50 μM cold dicamba, 2.5 μM [¹⁴C] dicamba(the final specific activity of the radioactive dicamba was 1.9mCi/mmol), and different amounts of cell lysate or partially purifiedenzyme. All enzyme assays during the final purification steps wereconducted in phosphate buffer because the pH optimum for dicambaO-demethylase activity was found to be in the mid range of phosphatebuffers, and higher enzyme activity was observed with phosphate buffercompared to Tris-HCl [tris(hydroxymethyl)aminomethane hydrochloride]buffer at pH 7.0. Reactions were initiated by the addition of thesubstrate, dicamba. At specific times, the reactions were stopped byadding 50 μl of 5% (vol/vol) H₂SO₄. Dicamba and dicamba metabolites werethen extracted twice with one volume of ether, and the extracts wereevaporated to dryness. The efficiencies of recovery (means±standarddeviations) for the extraction procedure were 87%±2% for dicamba and85%±3% for 3,6-DCSA (Yang et al., Anal. Biochem. 219:37-42 (1994)).

[¹⁴C]dicamba and ¹⁴C-labeled metabolites were separated by thin layerchromatography (TLC). The ether-extracted dicamba and its metaboliteswere redissolved in 50 μl of ether prior to being spotted onto a TLCplate. The solvent system for running the TLC waschloroform-ethanol-acetic acid (85:10:5 [vol/vol/vol]). The resolvedreaction products were visualized and quantified by exposing the TLCplate to a phosphor screen for 24 hours and then scanning the screen ina PhosphorImager SF (Molecular Dynamics, Sunnyvale, Calif.). Estimatesof the amount of radioactivity in a particular spot on the TLC platewere determined by comparing the total pixel count in that spot relativeto a spot on the same plate containing a known amount of [¹⁴C] dicamba.One unit of activity was defined as the amount of enzyme that catalyzesthe formation of 1 nmol of 3,6-DCSA from dicamba per minute at 30° C.Specific activities were based on the total protein concentration of theassay mixture.

The activity of the reductase component of dicamba demethylase wasassayed by measuring reduction of 2,6-dichlorophenolindophenol (DCIP)with a Hitachi U-2000 spectrophotometer. The reaction contained 0.5 mMNADH, 0.2 mM FAD (flavin adenine dinucleotide), 50 μM DCIP, 20 mM Trisbuffer (pH 8.0), and 10-100 μl of enzyme sample in a total volume of 1ml. The change in absorbance at 600 nm with time was measured at roomtemperature. Specific activity was calculated using an extinctioncoefficient at 600 nm of 21.0 mM⁻¹cm¹ for reduced DCIP. Specificactivity was expressed as nmol DCIP reduced min⁻¹ mg⁻¹ of protein.

In addition, an in situ DCIP assay was used to detect and locate thereductase activity in protein preparations separated on isoelectricfocusing (IEF) gels. After electrophoresis of the proteins on an IEFgel, lanes sliced from the gel were washed with 20 ml of cold 20 mMTris-HCl buffer (pH 8.0). Low melting agarose was dissolved by heatingin 10 ml of 20 mM Tris-HCl buffer (pH 8.0) at a final concentration of1.5% (w/v). When the agarose cooled to near room temperature, it wassupplemented with 0.2 mM FAD, 50 μM DCIP, and 0.5 mM NADH. The assaymixture was poured onto a glass plate and allowed to solidify. The gelpiece was placed on top of the solidified reaction mixture and allowedto set at room temperature for 15 minutes. If the gel slice contained aprotein with reductase activity, a colorless band of reduced DCIP wasgenerated in the blue background of DCIP.

Cell Lysates.

Cells were grown to an optical density at 550 nm of 1.3 to 1.5 in liquidreduced chloride medium containing a mixture of glucose and CasaminoAcids on a rotary shaker (250 rpm at 30° C.). The cells were harvestedby centrifugation, washed twice with cold 100 mM MgCl₂, and centrifugedagain. Cell pastes were either used immediately or quickly frozen inliquid nitrogen and stored at −80° C. At the time of enzymepurification, 25 g of frozen cells were thawed and resuspended in 50 mlof isolation buffer containing 25 mM Tris buffer (pH 7.0), 10 mM MgCl₂,and 0.5 mM EDTA. Phenylmethylsulfonyl fluoride and dithiothreitol wereadded to final concentrations of 0.5 mM and 1 mM, respectively. Afteraddition of 10 mg of lysozyme and 1 mg of DNase, cells were stirred for10 min on ice and broken by sonication (model XL2020 sonicator; HeatSystems) on ice at a medium setting (setting 5) with twelve 20-secondbursts and 40-second resting intervals. The resulting cell lysates werediluted to 90 ml with isolation buffer and centrifuged at 76,000×g for 1h at 4° C. The supernatant was used as the source of cleared celllysate.

Enzyme Purification.

All procedures were performed at 4° C., unless otherwise stated. Solidammonium sulfate was slowly added to a 90-ml volume of cleared celllysate to 40% (wt/vol) saturation, with constant stirring. After 15minutes of stifling, the mixtures were centrifuged at 15,400×g for 15minutes, and the precipitate was discarded. Additional solid ammoniumsulfate was added to 70% (wt/vol) saturation, with constant stifling ofthe supernatant. After 15 min of stirring, the mixtures were centrifugedunder the conditions described above. The supernatant was discarded, andthe precipitate was resuspended in a minimal volume of buffer A (20 mMTris [pH 8.0], 2.5 mM MgCl₂, 0.5 mM EDTA, 5% (vol/vol) glycerol, and 1mM dithiothreitol).

The 40%-70% ammonium sulfate cut was then loaded onto a Phenyl-Sepharosecolumn (2.5 by 10 cm) connected to a FPLC apparatus (Pharmacia) andeluted with a decreasing linear gradient of (NH₄)₂SO₄ from 10% (w/v) to0% (w/v). The column was preequilibrated with buffer A containing 10%(wt/vol) ammonium sulfate. The flow rate was 1 ml/min. Proteinconcentrations were continuously monitored at A₂₈₀ during columnelution. The column was washed with 120 ml of buffer A containing 10%(wt/vol) ammonium sulfate until baseline A₂₈₀ readings were obtained.Bound proteins were eluted with a decreasing gradient of (NH₄)₂SO₄ inbuffer A [10 to 0% (wt/vol) (NH₄)₂SO₄ in a total volume of 210 ml].Fractions of 2 ml were collected. Aliquots of 10 μl were taken from eachfraction and added to the standard dicamba O-demethylase assay mixture(see above), except that nonradioactive dicamba was used as thesubstrate. Dicamba O-demethylase activity was detected by monitoring theconversion of dicamba to the highly fluorescent reaction product3,6-DCSA with a hand-held UV lamp (312 nm, Fotodyne) in a darkened room.

This procedure allowed resolution of dicamba O-demethylase into threepools containing the separated components (designated components I, IIand III). Each component was essential for dicamba O-demethylaseactivity (see below). When a single component was assayed, the other twocomponents were provided in excess. Fractions containing a single typeof activity were pooled (component I, fractions 128-145; component II,unbound fractions 12-33; component III, fractions 62-92).

(i) Purification of Component I.

Fractions containing component I activity (eluting from aPhenyl-Sepharose column at 0 M (NH₄)₂SO₄, fractions 128-145) were pooledto provide a total volume of 34 ml. The pooled samples were concentratedto 10 ml by centrifugation in a Centriprep-10 device (Amicon) and thenapplied to a Q-Sepharose (Fast Flow) FPLC column (Pharmacia) (2.5 by 6cm) equilibrated with buffer A and washed with 80 ml of buffer A.Proteins bound to the column were eluted with a 100 ml linear gradientof 0 to 0.6 M KCl in buffer A at a flow rate of 1 ml/min. Fractions werecollected at 1.5 minute intervals. Those fractions exhibiting componentI activity (fractions 29-37) were pooled, dialyzed against buffer Aovernight at 4° C. and applied to a Mono Q HR 5/5 FPLC anion-exchangecolumn in buffer A. Proteins were eluted at 1 ml/min by using a 50 mlgradient of increasing KCl concentration (0 to 0.5 M). Fractions showingcomponent I activity (fractions 19 to 25) were pooled and concentratedto 0.4 ml by centrifugation in a Centricon-10 device. The concentratedsample was then subjected to chromatography on a Superose 12 FPLC column(1.6 by 50 cm) at a flow rate of 0.2 ml/min with buffer A containing 100mM KCl. Fractions 7-10 showing component I activity were pooled andconcentrated by centrifugation in a Centricon-10 device.

The partially purified component I was diluted with cold 1% (w/v)glycine and concentrated by centrifugation in a Centricon-10 devicethree more times to desalt it in preparation for IEF electrophoresis.The desalted and concentrated sample was then applied to a 6% (w/v) IEF(pH 4-6) gel and subjected to electrophoresis for 1.5 hours at 4° C.(see below). After electrophoresis, the gel was washed with 25 mM coldphosphate buffer (pH 7.0) for 5 minutes and then each slice of the gellane was diced into small (6 mm×4 mm) pieces. Protein was eluted fromthe diced gel fragments by grinding them with a pipette tip in thepresence of 10 μl of 25 mM phosphate buffer (pH 7.0). Protein from eachsegment was mixed with an excess of components II and III and assayedfor dicamba O-demethylase activity. The gel segment which showedcomponent I activity (which was also reddish brown in color) was loadedonto a 12.5% (w/v) sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE)to check sample purity.

(ii) Purification of Component II.

Component II obtained by Phenyl-Sepharose column chromatography wasdialyzed against buffer A overnight at 4° C. and applied to a FPLCQ-Sepharose column (2.5 by 6 cm). Sample elution conditions wereidentical to those described above for component I except that theelution gradient was 0 to 1 M KCl in buffer A. Fractions exhibitingcomponent II activity (fractions 30-37) were pooled, dialyzed againstbuffer A, concentrated to 0.4 ml and applied to a FPLC Superose 12column (1.6 by 50 cm). The procedures for sample application and elutionwere identical to those described above for component I. Fractionsexhibiting component II activity (fractions 3-6) were pooled, dilutedwith an equal volume of buffer A, and applied to a FPLC Mono Q column.Proteins were eluted from the column using the same KCl gradient as forcomponent I. Fractions 20-25 showed component II activity. Partiallypurified component II was further purified by IEF (pH 4-6)electrophoresis using the same conditions as described for component I.The gel segment which showed component II activity was loaded onto a12.5% (w/v) SDS-PAGE for further analysis.

(iii) Purification of Component III.

Component III obtained by Phenyl-Sepharose column chromatography wasdialyzed against buffer A overnight at 4° C. and applied to a FPLCQ-Sepharose column (2.5 by 6 cm). Conditions were identical to thosedescribed above for component I. Fractions exhibiting component IIIactivity (fractions 26-38) were dialyzed against buffer B [10 mMTris-HCl (pH 7.5), 2.5 mM MgCl₂, 5% (v/v) glycerol, 1 mM dithiothreitol]and concentrated to 5 ml. Blue dye affinity matrix [Cibacron Blue 3GAtype 3000 (Sigma)] was packed into a FPLC column (1×5 cm) andpre-equilibrated with 20 ml of buffer B. Concentrated component III wasloaded onto the blue dye column and washed with 20 ml of buffer B at aflow rate of 0.2 ml/min until the A₂₈₀ of the column effluent reachedbaseline levels. Bound protein was then eluted with 5 mM NADH in bufferB. Fractions containing reductase activity were detected by assaying fordicamba O-demethylase activity in the presence of an excess ofcomponents I and II and also by the ability of each fraction to reduceDCIP in the presence of NADH. Fractions having strong reductase activityin both assays were pooled, dialyzed against buffer A containing 100 mMKCl, concentrated to 0.2 ml, and applied to a FPLC Superose 12 column.The same conditions were used for running the Superose column asdescribed for component I. Fractions containing proteins which catalyzedDCIP reduction were pooled, dialyzed against buffer A and applied to aFPLC Mono Q column. Proteins were eluted using the same conditions asfor component I. Partially purified component III was further purifiedby IEF (pH 4-6) gel electrophoresis. The reductase activity of proteinswithin the IEF gel was detected by assaying for DCIP reduction in anagarose gel overlay as described above. The gel segment which showedcomponent II activity was loaded onto a 12.5% (w/v) SDS-PAGE for furtheranalysis.

Determination of NH₂-Terminal Amino Acid Sequences.

Protein bands were excised from IEF gels and placed in the wells of a12.5% (w/v) SDS polyacrylamide gel. After electrophoresis, the gelslices containing the purified proteins were transblotted onto a PVDF(polyvinylidene difluoride) membrane (Millipore) in a Trans-Blot cell(Bio-Rad, Richmond, Calif.) at 25 volts for 16 hours. The blottingbuffer was a solution of 20% (v/v) methanol with 10 mM CAPS[3-(cyclohexylamino)-1-propanesulfonic acid], pH 10.0. Sequencing wasperformed using an Applied Biosystems Inc. 420 H machine by Edmandegradation (Edman and Henschen (1975) pages 232-279 in S. B. Needleman(ed.), Protein sequence determination, 2nd ed., Springer-Verlage, NewYork).

Determination of Protein Concentration.

Protein concentrations were determined by the method of Bradford (1976)Anal. Biochem. 72:248-254, with bovine serum albumin as the standard.

SDS-PAGE.

Sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) wasperformed according to modified methods of Laemmli (Laemmli (1970)Nature, 227:680-685). 12.5% (w/v) SDS gels of 85×65×0.75 mm were made asfollows: running gel: 2.5 ml 40% (w/v) acrylamide/bis solution (37:5:1),1 ml running buffer solution [3M Tris-HCl (pH 8.8), 0.8% (w/v) SDS], 4.5ml H₂O, 5 μl TEMED, and 40 μl 10% (w/v) APS; stacking gel: 0.5 ml 40%(w/v) acrylamide/bis, 0.5 ml stacking buffer solution [1 M Tris-HCl (pH6.8), 0.8% (w/v) SDS], 3 ml H₂O, 5 μl TEMED, and 12.5 μl 10% (w/v) APS.The composition of the running buffer was 25 mM Tris-HCl (pH 8.3), 0.2 Mglycine, and 0.1% (w/v) SDS. The sample buffer contained 0.25 mlstacking buffer, 0.6 ml 20% (w/v) SDS, 0.2 ml β-mercaptoethanol, and0.95 ml 0.1% bromphenol blue (w/v) in 50% (v/v) glycerol.Electrophoresis was performed at 80 volts in a Bio-Rad Mini Gelapparatus until the tracking dye was 0.5 cm from the anode end of thegel. Proteins were stained with 0.1% (w/v) Coomassie Brilliant BlueR-250 in a mixture of isopropanol, water, and acetic acid at a ratio of3:6:1 (v/v/v). Destaining was performed in a mixture of methanol, water,and acetic acid at a ratio of 7:83:10 (v/v/v). Standard proteins (GibcoBRL) included: myosin (214.2 kDa), phosphorylase B (111.4 kDa), bovineserum albumin (74.25 kDa), ovalbumin (45.5 kDa), carbonic anhydrase(29.5 kDa), β-lactoglobulin (18.3 kDa), and lysozyme (15.4 kDa).

Determination of Molecular Weight.

The molecular weight (M_(r)) of peptides under denaturing conditions wasestimated using SDS-PAGE analysis. The molecular weights of the nativecomponents I, II and III were estimated by gel filtration through aSuperose 12 HR 10/30 FPLC column (Pharmacia) at a flow rate of 0.2ml/min in buffer A containing 100 mM KCl. Calibration proteins were gelfiltration standards from Bio-Rad. They were: bovine thyroglobulin (670kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), horsemyoglobin (17 kDa) and vitamin B-12 (1.35 kDa). The void volume of theSuperose 12 column was calculated using Blue Dextran (M_(r) 2,000,000,Sigma).

IEF.

Isoelectric focusing (IEF) gel electrophoresis was performed in avertical mini-gel apparatus (Model #MGV-100) from C.B.S. Scientific Co.(Del Mar, Calif.). IEF gels with 6% (w/v) polyacrylamide (70×90×1 mm)were made by mixing the following: 1.6 ml 30% (w/v) acrylamide/bis(37:5:1), 0.8 g glycerol, 0.32 ml ampholyte pH 4-6 (Serva), 0.08 mlampholyte pH 4-9 (Serva), 5.2 ml H₂O, 10 μl TEMED, and 80 μl 10% (w/v)APS. The cathode buffer was 100 mM β-alanine and the anode buffer was100 mM acetic acid. Protein samples in approximately 1 to 10 μl of 1%(w/v) glycine were mixed with an equal volume of sample buffer [50%(v/v) glycerol, 1.6% (v/v) ampholyte pH 4-9, 2.4% (v/v) ampholyte pH4-6]. Samples were loaded at the cathode end of the gel and allowed tomigrate at 200 volts for 1.5 hours and 400 volts for another 1.5 hours.Proteins were stained with Coomassie Brilliant Blue R-250 using theprocedure described above for SDS polyacrylamide gels. IEF markers(Sigma) were: amyloglucosodase, pI 3.6; glucose oxidase, pI 4.2; trypsininhibitor, pI 4.6; β-lactoglobulin A, pI 5.1; carbonic anhydrase II, pI5.4; carbonic anhydrase II, pI 5.9 and carbonic anhydrase I, pI 6.6

Kinetic Analysis.

The kinetics of the demethylation reaction catalyzed by dicambaO-demethylase were studied by analyzing the initial rates of thereaction in the presence of a constant concentration of the enzyme andincreasing concentrations of the substrate, dicamba. Reaction mixturescontained 25 mM potassium phosphate buffer (pH 7.0), 10 mM MgCl₂, 0.5 mMNADH, 0.5 mM FeSO₄, 25 μg of partially purified O-demethylase enzyme[the 40%-70% (w/v) (NH₄)₂SO₄ fraction from a cleared cell lysate],various concentrations (0.5 to 50 μM) of dicamba and variousconcentrations (0.025 to 2.5 μM) of [¹⁴C]dicamba (U-phenyl-¹⁴C, 42.4mCi/mmol) in a total volume of 300 μl For assays with dicambaconcentrations of 0.5 μM and 1 μM, the reaction volume was increased to900 μl to ensure that sufficient amounts of radioactive dicamba and itsmetabolites were present to allow detection. In these reactions, theamounts of all other components in the reaction were tripled. Theconversion of [¹⁴C]dicamba to [¹⁴C]3,6-DCSA was determined for differenttime points at each concentration of dicamba using a PhosphorImager SFto scan radioactivity on phosphor screens which had been exposed to TLCplates for 24 hours. One unit of activity was defined as the amount ofenzyme that forms 1 nmol of 3,6-DCSA per minute at 30° C. The initialrates of each reaction were determined by plotting the reaction rateversus time at each substrate concentration. Data were modeled toMichaelis-Menten kinetics and values of K_(m) and V_(max) weredetermined by fitting to Lineweaver-Burk plots using SigmaPlot® (JandelScientific, Corte Madera, Calif.).

Oxygen Requirement.

Preliminary experiments using a Clark oxygen electrode indicated oxygenconsumption during a standard dicamba O-demethylase assay with dicambaas a substrate. To verify a requirement for oxygen in the Odemethylation of dicamba by dicamba O-demethylase, reactions wereconducted in an anaerobic chamber which contained less than 1 ppm ofoxygen. The procedures for displacement of oxygen from the reactionmixture were performed at 4° C. Reaction mixtures lacking enzyme wereplaced in a vial and sealed with a rubber stopper. For displacement ofoxygen, the vial was evacuated twice by vacuum and flushed each timewith nitrogen. After a third evacuation, the vial was flushed with 90%nitrogen plus 10% hydrogen. The enzyme solution was likewise purged ofoxygen (with care taken not to bubble the enzyme solution). Both thereaction mixtures and enzyme solutions were transferred into ananaerobic chamber (95% N₂-5% H₂ atmosphere). Two hundred fortymicroliters of cleared cell lysate was injected through the rubberstopper with a microsyringe and gently mixed with 960 μl of oxygen-freereaction mixture. Reactions were carried out at 30° C.

An examination of the reaction products on TLC plates showed that therate of [¹⁴C]3,6-DCSA production from [¹⁴C]dicamba under anaerobicconditions was significantly lower than the rate of reactions with thesame amount of enzyme under aerobic conditions. Under anaerobicconditions, there was virtually no conversion of dicamba to 3,6-DCSAwithin 1 hour. However, when a parallel reaction mixture was taken fromthe anaerobic chamber after 30 min and incubated with air, a significantquantity of one of the components of the dicamba O-demethylase enzymecomplex was an oxygenase.

It may be noted that the in vitro conversion of [¹⁴C]dicamba to[¹⁴C]3,6-DCSA mimics the in vivo conversion pathway documented earlier(Cork and Kreuger, Adv. Appl. Microbiol. 36:1-66 (1991); Yang et al.,Anal. Biochem. 219:37-42 (1994)). In these studies, DCSA was identifiedas a reaction product by both TLC and capillary electrophoresis.Stringent identification of the first major product of dicambadegradation as DCSA both in vivo and in vitro has been obtained by gaschromatography-mass spectrometry analyses.

Component and Cofactor Requirements.

After the initial separation of the three components of dicambaO-demethylase by phenyl-Sepharose column chromatography, the partiallypurified preparations were taken individually through one additionalpurification on a Q-Sepharose column (2.5 by 6 cm). Samples were appliedto a Q-Sepharose (Fast Flow) fast protein liquid chromatography column(Pharmacia) in buffer A and eluted with a 100-ml linear gradient of 0 to0.6 M KCl (for the oxygenase component) or 0 to 1.0 M KCl (for theferredoxin and reductase components) in 1.5-ml fractions. Appropriatepooled fractions from separate columns for oxygenase purification(fractions 29 to 37), for ferredoxin purification (fractions 30 to 37),or for reductase purification (fractions 26 to 38) were used for thedetermination of component and cofactor requirements.

The three components were assayed for O-demethylase activity in variouscombinations to determine component requirements.

To determine cofactor requirements, O-demethylase activity was assayedusing a mixture of the three components with [¹⁴C]dicamba for 30 minutesat 30° C. The amounts of partially purified protein (provided in anoptimized ratio) in the reaction mixtures were 85 μg of oxygenase, 55 μgof ferredoxin and 50 μg of reductase. The concentration of cofactorsused in the reaction mixtures were 0.5 mM NADH, 0.2 mM FAD, 0.5 mMFeSO₄, 10 mM MgCl₂, 0.5 mM NADPH, and 0.2 mM FMN.

Results

Component I.

The component of dicamba O-demethylase which bound most tightly to thePhenyl-Sepharose column (designated initially as component I andsubsequently identified as an oxygenase) was distinctly reddish brown incolor. This indicated the potential presence of a protein(s) containingan iron-sulfur cluster(s) or a heme group(s). The fractions withcomponent I activity from the Phenyl-Sepharose column were subjected tofurther purification by Q-Sepharose (Fast Flow) and Mono Qchromatography and then to separation on a Superose 12 size exclusioncolumn. The component I protein was then further purified on an IEF gel.

Protein from the major band on the IEF gel (with an apparent pI ofapproximately 4.6) was excised and separated from any remaining minorcontaminants by SDS-PAGE. The major component I protein obtained afterpurification by IEF was greater than 90% pure as judged by densitometricanalysis of this SDS-polyacrylamide gel stained with Coomassie Blue.

The N-terminal amino acid sequence of the dominant protein with anapparent molecular mass of approximately 40,000 Daltons was determined.Results of amino acid sequencing indicated that the first 29 amino acidsof the N-terminal region were present in the following sequence(residues in parentheses are best guesses):

[SEQ ID NO: 1] Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp (Asp or Thr) (Pro).Comparison with amino acid sequences in various databases indicatedlittle or no homology with NF₂-terminal sequences reported for othermonoxygenases or dioxygenases.

Component II.

The protein fraction which did not bind to a Phenyl-Sepharose column wasdesignated as component II. Because this yellowish colored fractioncould be replaced by ferredoxin from Clostridium pasteurianum (but withslower reaction rates) when assays were performed in combination withcomponents I and III, it was tentatively designated as aferredoxin-containing fraction. The Clostridium ferredoxin clearlyfunctioned in place of component II, but given the highly impure natureof the component II used in these experiments, the efficiency of theClostridium enzyme was significantly lower than that of the putativeferredoxin from strain DI-6. In particular, 55 μg of partially purifiedcomponent II mixed with excess amounts of components I and III catalyzedthe conversion of dicamba to 3,6-DCSA at a rate of approximately 5 nmolmin⁻¹ mg⁻¹, while 100 μg of highly purified ferredoxin from Clostridiumresulted in an activity of only 0.6 nmol min⁻¹ mg⁻¹.

Purification steps involving Q-Sepharose (Fast Flow) chromatography,Superose 12 gel filtration and Mono Q chromatography yieldedapproximately one milligram of purified protein from an initial 25 gramsof cell paste. This fraction was purified in a similar manner to theoxygenase component by electrophoresis on an IEF gel and subsequentelectrophoresis of the active IEF fraction on an SDS-polyacrylamide gel.

Analysis of component II activity in proteins eluted from segments ofthe IEF gel indicated that a fraction with a pI of approximately 3.0contained the active protein in component II. Protein from this gelslice was eluted and subjected to SDS-PAGE. Staining of the gel withCoomassie Blue revealed one prominent band of protein (molecular weightof about 28,000 Daltons) along with a smear of lower molecular weightproteins.

Component III.

Component III of dicamba O-demethylase was retained on aPhenyl-Sepharose column in high concentrations of (NH₄)₂SO₄ and elutedat approximately 4% (w/v) (NH₄)₂SO₄. This light yellow fraction wastentatively identified as a reductase-containing fraction based on itsability to reduce oxidized cytochrome c and DCIP in the presence of NADHand because it could be replaced by cytochrome c reductase from porcineheart (Type 1, Sigma) in assays with components I and II. In this set ofreactions, the use of 50 μg of partially purified component III produceda reaction rate of approximately 5 nmol min⁻¹ mg⁻¹ when mixed with anexcess of components I and II. The highly purified cytochrome creductase showed a specific activity of approximately 2.5 nmol min⁻¹mg⁻¹ in the reaction, an activity well below that provided by componentIII when one considers the impurity of the crude component III used inthese assays. In addition, component III exhibited reductase activitywhen incubated with cytochrome c or 2,6-dichlorophenol-indophenol(DCPIP) in the presence of NADH. Neither component I nor component IIshowed activity in either of these two reductase assays.

Additional purification of this fraction by chromatography on columnscontaining Q-Sepharose (Fast Flow), blue dye affinity matrix, Superose12, and Mono Q packings resulted in low amounts of protein in thefractions with reductase activity. The component III protein was about70% pure as judged by densitometric analysis of the active proteinfraction after separation by SDS-PAGE and staining with Coomassie Blue.

To further exacerbate purification of component III, it was found thattwo different protein fractions from the Mono Q column step containedreductase activity when assayed with the ferredoxin and oxygenasecomponents. Further purification of these two fractions byeletrophoresis on an IEF gel revealed that the reductase activities ofthe two fractions had distinctly different isoelectric points. This wasdemonstrated by excising lanes containing each of the two reductasefractions from the IEF gel and laying the slices on top of a pad of lowmelt agarose containing a DCIP reaction mixture. Reductase activity inboth gel slices was identified by the NADH-dependent reduction of DCIPto its colorless, reduced form. The reductase in fraction 35 had anapparent pI of approximately 5.6 while the reductase in fraction 27possessed an apparent pI of approximately 4.8.

Both reductase activities isolated from the IEF gel slices were unstableand present in low amounts. Indeed, only the reductase from fraction 35from the Mono Q column fractionation retained sufficient proteinconcentration and activity to allow further purification andcharacterization. A slice from an IEF gel containing this reductaseactivity was eluted and separated from contaminating proteins bySDS-PAGE. The predominant protein in this gel was one with a mass ofapproximately 45,000 Daltons. Size exclusion chromatography hadindicated an approximate molecular mass of 50,000 Daltons for componentIII in its native state.

Biochemical Characteristics of Dicamba O-Demethylase.

Dicamba O-demethylase activity was measured during incubations in vitroat temperatures ranging from 20° C. to 50° C. and at pH values fromapproximately 6 to 9. Activity peaked sharply at 30° C. and broadly atpH values between 6.5 and 7.5. Enzymatic activity was dependent on thetype of pH buffer employed. At pH 7, for example, activity wasapproximately 40% lower in Tris-containing buffers than inphosphate-containing buffers.

Values for K_(m) and V_(max) for dicamba O-demethylase were estimatedusing SigmaPlot® to generate best fit curves from Michaelis-Menten andLineweaver-Burk plots of data from duplicate experiments. The K_(m) fordicamba was estimated to be approximately 9.9±3.9 μM and the V_(max) forthe reaction was estimated to be approximately 108±12 nmol/min/mg.

The three components were assayed for dicamba O-demethylase activity invarious combinations. None of the components showed enzyme activity whenassayed alone. Indeed, a significant amount of O-demethylase activitycould be detected only when all three components were combined. Amixture of components I and II exhibited small amounts of enzymeactivity, probably due to traces of component III contaminating thecomponent I fractions.

Both NADH and NADPH supported enzyme activity, with NADH being markedlymore effective than NADPH. Mg²⁺ was necessary for enzyme activity. Fe²⁺,flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN)produced little or no stimulation of enzymatic activity with thepartially purified protein preparations in these experiments. Thehighest activity was obtained using a combination of NADH, Fe²⁺, Mg²⁺,and FAD.

Discussion

Dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6, is athree component oxygenase (Wang, X-Z (1996) Ph.D. thesis, University ofNebraska-Lincoln, Lincoln, Neb.) responsible for the conversion of theherbicide, dicamba (2-methoxy-3,6-dichlorobenzoic acid), to3,6-dichlorosalicylic acid (3,6-DCSA; 2-hydroxy-3,6-dichlorobenzoicacid). Purification schemes have been devised which have allowed theisolation of each of the three components to a homogeneous ornear-homogeneous state.

Initial separation of the three components was achieved bychromatography on a Phenyl-Sepharose column. Enzymatic activities andother characteristics of the partially purified components allowed atentative identification of the components as a reductase, a ferredoxinand an oxygenase—a composition similar to that found in a number ofother previously studied heme-containing and nonheme-containing,multicomponent oxygenases (Batie, et al. (1992) pages 543-565, In F.Müller (ed.), Chemistry and biochemistry of flavoenzymes, vol. III, CRCPress, Boca Raton; Harayama, et al. (1992) Annu. Rev. Microbiol.46:565-601; Mason and Cammack (1992) Annu. Rev. Microbiol. 46:277-305;Rosche et al. (1995) Biochem. Biophys. Acta 1252:177-179). Component IIIisolated from the Phenyl-Sepharose column catalyzed the NADH-dependentreduction of both cytochrome c and the dye, DCIP. In addition, itsability to support conversion of dicamba to 3,6-DCSA when combined withcomponents I and II could be replaced in part by cytochrome c reductase.Component II could be replaced by the addition of ferredoxin fromClostridium pasteurianum to reactions containing components I and III.The absolute need for molecular oxygen to support the O-demethylationreaction indicated that the remaining component was an oxygenase.

Oxygenase_(DIC).

Component I of dicamba O-demethylase (designated as oxygenase_(DIC)) hasbeen purified to homogeneity and subjected to N-terminal amino acidsequencing. The resulting sequence of twenty nine amino acid residuesshowed no significant homology to other protein sequences in the variousdata banks. However, the information obtained from this amino acidsequence permitted the design of degenerate oligonucleotide probes whichhave been successfully used to detect and clone the component I gene(see Example 2). Furthermore, a comparison of the amino acid sequencederived from the nucleotide sequence of this clone with that of theprotein sequences in the data base showed strong homology to otheroxygenases (see Example 2).

The apparent molecular mass of oxygenase_(DIC), estimated from itsmigration in SDS-polyacrylamide gels, is approximately 40,000 Daltons.Purified preparations of the oxygenase exhibited only one major band onSDS-polyacrylamide gels stained with Coomassie Blue and Edmandegradation of the protein contained in that band indicated the presenceof only one N-terminal species. Estimates derived from the behavior ofnative oxygenase_(DIC) on size exclusion columns suggests a molecularsize of approximately 90,000 Daltons. All of these results suggest thatthe native oxygenase exists as a homodimer.

The oxygenase/hydroxylase component of a number of multicomponentsystems is composed of an (αβ)_(n)-type subunit arrangement in which thelarger α subunit is approximately 50,000 Daltons in size and the smallerβsubunit is approximately 20,000 Daltons in molecular mass (Harayama, etal. (1992) Annu. Rev. Microbiol. 46:565-601). In contrast, the oxygenasecomponent of dicamba O-demethylase appears to possess a single subunitof approximately 40 kDa in molecular mass which may exist as a dimer inits native state. This (α)_(n)-type subunit arrangement is similar tothat found in other well characterized oxygenases such as4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas sp. strain CBS(Markus, et al. (1986) J. Biol. Chem. 261:12883-12888), phthalatedioxygenase from Pseudomonas cepacia (Batie, et al. (1987) J. Biol.Chem. 262:1510-1518), 4-sulphobenzoate 3,4-dioxygenase from Comamonastestosteroni (Locher, et al. (1991) Biochem. J., 274:833-842),2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86(Rosche et al. (1995) Biochem. Biophys. Acta 1252:177-179),4-carboxydiphenyl ether dioxygenase from Pseudomonas pseudoalcaligenes(Dehmel, et al. (1995) Arch. Microbiol. 163:35-41), and 3-chlorobenzoate3,4-dioxygenase from Pseudomonas putida, (Nakatsu, et al. (1995)Microbiology (Reading) 141:485-495).

Ferredoxin_(DIC).

Component II (ferredoxin_(DIC)) of dicamba O-demethylase was purified byseveral steps of column chromatography and IEF. Final purification bySDS-PAGE produced one major band of protein (M_(r)-28,000) and a smearof slightly smaller proteins.

The N-terminal amino acid sequence of the protein with an apparentmolecular weight of approximately 28,000 Daltons was determined. Thisamino acid sequence permitted the preparation of degenerateoligonucleotide probes, and these probes were used to isolate a genomicclone coding for this protein. Although the protein produced by thisclone was a ferredoxin (ferrodoxin_(28kDa)), it was subsequentlydetermined not to be active in the degradation of dicamba when combinedwith the other two components of dicamba O-demethylase (data not shown).

Other evidence supports the conclusion that ferrodoxin_(28kDa) is notthe ferredoxin component of dicamba O-demethylase. First, the molecularmass of this protein (28 kDa) protein is significantly higher than thatof the other ferredoxins found in multicomponent oxygenases frombacteria (i.e., 8-13 kDa) (Batie, et al. (1992) pages 543-565, In F.Müller (ed.), Chemistry and biochemistry of flavoenzymes, vol. III, CRCPress, Boca Raton; Harayama, et al. (1992) Annu. Rev. Microbiol.46:565-601).

Second, a comparison of the N-terminal sequence of 20 amino acidresidues obtained from ferredoxin_(28kDa) to other amino acid sequencesin the various protein data banks using Genetics Computing Group (GCG)software package (University of Wisconsin, Madison, Wis.) revealedstrong homology (80-85% identity compared to the most likely N-terminalsequence of ferredoxin_(28kDa)) to a number of dicluster bacterialferredoxins (those from Pseudomonas stutzeri, Pseudomonas putida,Rhodobacter capsulatus and Azotobacter vinelandii). The four diclusterferredoxins which showed strong homology to ferredoxin_(28kDa) have a[3Fe-4S] cluster followed by a [4Fe-4S] cluster at the N-terminus of theprotein. This suggests that ferredoxin_(28kDa) is distinctly differentfrom the ferredoxin components with [2Fe-2S] clusters which are usuallyassociated with non-heme multicomponent oxygenases (Harayama, et al.(1992) Annu. Rev. Microbiol. 46:565-601; Mason and Cammack (1992) Annu.Rev. Microbiol. 46:277-305; Rosche, et al. (1995) Biochem. Biophys. Acta1252:177-179).

Reductase_(DIC).

Component III of dicamba O-demethylase (designated as reductase_(DIC))has been the most recalcitrant of the three components to purify. Thisis due in part to its apparent instability and low abundance in lysatesof strain DI-6. Nonetheless, sufficient protein has been purified toassign a tentative molecular mass of 45,000 Daltons. This is similar tothe molecular mass of approximately 50,000 Daltons obtained from sizeexclusion chromatography and suggests that reductase_(DIC) exists in itsnative form as a monomer. The purification of the reductase componenthas been further complicated by the fact that chromatography on a Mono Qcolumn and IEF resolves purified reductase preparations into twoactivities with apparently distinct pI values. Both fractions from theMono Q column functioned in combination with purified ferredoxin_(DIC)and oxygenase_(DIC) to produce dicamba O-demethylase activity. Thepresence in Sphingomonas sp. strain RW1 of two similar flavoproteinswhich function equally well as reductase components in the threecomponent dibenzofuran 4,4a-dioxygenase has recently been reported byBünz and Cook (Bünz and Cook (1993) J. Bacteriol. 175:6467-6475).Interestingly, both reductases were 44,000 Daltons in molecular mass,quite similar to that of the 45,000 Dalton reductase_(DIC). Multiplecomponents of leghemoglobin reductase have also been observed in lupinroot nodules using isoelectric focusing techniques (Topunov, et al.(1982) Biokhimiya (English edition) 162:378-379). In this case, IEFrevealed four separate components with NADH-dependent reductaseactivity. The resolution of the question of whether there is only onereductase_(DIC) which exists in two forms or two distinct reductases instrain DI-6 will rely on the development of improved procedures forisolating larger quantities of the proteins and/or on the cloning andsequencing of the gene(s) involved (see Examples 3 and 5).

Dicamba O-Demethylase Characteristics.

In addition to the physical and biochemical properties of the individualcomponents noted above, analyses of enzymatic activity have shown thatthe O-demethylase system has a strong affinity (K_(m)=−10 μM) for itssubstrate and a V_(max) of approximately 100-110 nmol/min/mg. Asexpected for a soil bacterium collected in a temperate climatic zone,the maximal enzymatic activity was observed at temperatures near 30° C.While the pH optima for the enzyme system was in the range from pH 6.5to pH 7.5, the activity measured with a given preparation of enzyme wasstrongly affected by the type of pH buffering system employed. Activityin the presence of Tris buffers was at least 40% lower than withphosphate buffers at the same pH.

The reaction scheme for the reaction catalyzed by the three componentsof dicamba O-demethylase is presented in FIG. 1. Electrons from NADH areshuttled through a short electron chain consisting of the reductase andferredoxin to the terminal oxygenase which catalyzes the oxidation ofdicamba. The similarities between dicamba O-demethylase and severalmulticomponent dioxygenases suggest that dicamba O-demethylase maypotentially possess cryptic dioxygenase activity. It is clear, however,that this enzyme is not in the class of dioxygenases which split O₂ andincorporate one atom of oxygen into the major substrate and the otherinto a small organic substrate such as α-ketoglutarate (Fukumori andHausinger (1993) J. Biol. Chem. 268:24311-24317). Indeed, combinationsof highly purified reductase_(DIC), ferredoxin_(DIC), andoxygenase_(DIC) require only O₂, NADH, Mg²⁺, Fe²⁺, and dicamba foractivity.

Example 2 Identification and Sequencing of a Clone Coding for theOxygenase of Dicamba O-Demethylase of Pseudomonas maltophilia DI-6

As noted in Example 1, the first 29 amino acids of the N-terminal aminoacid sequence of oxygenase_(DIC) had been determined to be (residues inparentheses are best guesses):

[SEQ ID NO: 1] Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu Glu Leu Ser Glu Lys Pro Leu Gly Arg ThrIle Leu Asp (Asp or Thr) (Pro).

This sequence permitted the design of degenerate oligonucleotide probeswhich were synthesized by Operon, Alameda, Calif. In particular, amixture of 32 probes, each of which was 17 nucleotides in length, andcontained all of the possible nucleotide sequences which could encodethe amino acid sequence highlighted in bold above, was used. Theoligonucleotide probes were 3′-end-labeled with digoxigenin (DIG)according to instructions provided by Boehringer Mannheim, Indianapolis,Ind.

The DIG-labeled probes were first hybridized to P. maltophilia DI-6genomic DNA which had been digested with various combinations ofrestriction enzymes, resolved on a 1% agarose gel, and blotted to anylon filter. Based on these results, a size-fractionated genomiclibrary was constructed in the pBluescript II KS+ vector and transformedinto Escherichia coli DH5α competent cells. The genomic librarycontained 1-2 kb Xho I/Hind III fragments. The DIG-labeledoligonucleotide probes were hybridized to an array of bacterial coloniesstreaked on nylon filters. Plasmid DNA was isolated from positivecolonies and subcloned. Both strands of each subclone were sequenced bythe DNA Sequencing Facility at the University of Nebraska-Lincoln.Hybridization and detection of DIG-labeled probes were performedaccording to protocols provided by Boehringer Mannheim.

A genomic DNA clone coding for the oxygenase_(DIC) was identified. Thenucleotide sequence and the deduced amino acid sequence of the entireoxygenase_(DIC) are given in the Sequence Listing below as SEQ ID NO:2and SEQ ID NO:3, respectively.

A comparison of the amino acid sequence derived from the nucleotidesequence of this clone with that of the protein sequences in the SwissProtein Database showed homology to other oxygenases. Homology wasdetermined using the FASTA program of the GCG software package. Thestrongest homology was with the oxygenase component of vanillatedemethylase (from Pseudomonas sp., ATCC strain 19151) which showed 33.8%identity.

Example 3 Purification and Characterization of Components of DicambaO-Demethylase of Pseudomonas maltophilia DI-6

Bacterial Cultures and Preparation of Cleared Cell Lysates.

Pseudomonas maltophilia, strain DI-6, was inoculated into six two-literErlenmeyer flasks containing one liter of reduced chloride medium(Kreuger, J. P. 1989. Ph.D. thesis. Illinois Institute of Technology,Chicago) supplemented with glucose (2.0 mg/ml) and Casamino acids (2.0mg/ml) as the carbon sources. Cultures were incubated on an orbitalshaker (225 rpm at 30° C.). Cells were harvested at an A₆₀₀ ranging from1.5 to 2.0 using a JLA-10.500 rotor in a Beckman Avanti J-25I Centrifugeat 4,000×g for 20 minutes. Pelleted cells were stored at −80° C. Thefrozen cells were resuspended in 40 ml of 100 mM MgCl₂, pelleted againusing the same conditions as above, and then resuspended in breakingbuffer (100 mM 3-[N-morpholino]propanesulfonic acid (MOPS) (pH 7.2), 1mM dithiothreitol, 5% glycerol) in a ratio of 2 ml breaking buffer pergram of cells wet weight. Lysozyme was added in a ratio of 80 μl pergram of cells along with a Protease Inhibitor Cocktail for bacterialextracts (Sigma, P 8465) in a ratio of 5 ml per 20 grams of cells wetweight. Finally, phenylmethylsulfonyl fluoride (0.1 M stock solution in100% ethanol) was added in a ratio of 250 μl per 50 ml of breakingbuffer. The cells were disrupted with a sonicator (Sonics and MaterialsInc., Model VCX 600) in 9.0 second bursts, with 3.0 second restingperiods, for 30 minutes at an amplitude of 50%. Lysed cells werecentrifuged for 75 minutes at 56,000×g in a JA-25.50 rotor of a BeckmanAvanti J-25I Centrifuge at 4° C. The supernatant (cleared cell lysate)was decanted and glycerol was added to a final concentration of 15%prior to storage at −80° C.

Initial Purification of Dicamba O-Demethylase Components.

An aliquot of the cleared cell lysate containing approximately 2.7 gramsof protein was applied to a Pharmacia XK 26/60 column containing 25 mlof DEAE-Sepharose Fast Flow equilibrated with 50 mM MOPS (pH7.2), 1 mMdithiothreitol, and 15% (v/v) glycerol (buffer A). The column wasconnected to a Bio-CAD Perfusion Chromatography Workstation (USDA NRICGPGrant #9504266) and run at a flow rate of 5.0 ml/min. After the columnwas loaded, it was washed with buffer A until the absorbance reading at280 nm decreased to below 0.1. All three components of dicambaO-demethylase were bound to the DEAE column under these conditions. Thecolumn was developed with a linear gradient of 0 to 500 mM NaCl inbuffer A. This resulted in the elution of the ferredoxin at 400 mM NaCland the co-elution of the reductase and oxygenase components at 250 mMNaCl.

Purification of the Ferredoxin_(DIC).

Fractions containing the ferredoxin_(DIC) eluted from the DEAE-Sepharosecolumn were pooled and buffer exchanged into 50 mM MOPS (pH 7.2), 5%glycerol (v/v) and 200 mM NaCl (buffer B). They were then concentratedto approximately 2 ml using a Amicon Cell Concentrator with a YM10membrane and a Centricon 10 concentrator. This sample was then appliedto a pre-packed Pharmacia HiPrep 26/60 Sephacryl S-100 columnequilibrated with buffer B and run at a flow rate of 0.5 ml/min on aPharmacia FPLC apparatus. Fractions that showed activity were pooled andbuffer exchanged into 50 mM MOPS (pH 7.2), 1 mM dithiothreitol and 5%glycerol (v/v) (buffer C). The fractions were concentrated toapproximately 2 ml and then loaded onto a Pharmacia Mono Q HR 5/5 columnequilibrated in buffer C. The column was developed with a lineargradient of 0-2.0 M NaCl in buffer C. Fractions that containedferredoxin activity were assayed for protein content and stored at −80°C.

Purification of the Reductase_(DIC).

Fractions from the initial DEAE-Sepharose column containing theoxygenase/reductase components were pooled, and ammonium sulfate wasadded to a final concentration of 1.5 M. After incubating at 4° C. for1.5 hours, the samples were centrifuged for 75 minutes at 56,000×g at 4°C. in a JA-25.50 rotor of a Beckman Avanti J-25I centrifuge. Thesupernatant was retained and loaded at a flow rate of 5.0 ml/min onto aPharmacia XK 26/20 column containing 25 ml of Phenyl-Sepharose 6 FastFlow (high sub) that was equilibrated in buffer A containing 1.5 M(NH₄)₂SO₄. Fractions that contained the reductase component were pooled,buffer exchanged into buffer B, and concentrated to approximately 2 mlusing the Amicon concentrator with an YM30 membrane and a Centricon 10concentrator. The 2 ml sample was applied to a pre-packed PharmaciaHiPrep 26/60 Sephacryl S-100 column equilibrated in buffer B and run at0.5 ml/min. Fractions containing reductase activity were pooled, bufferexchanged into buffer C, and concentrated down to approximately 2 ml.The 2 ml sample was loaded onto a pre-packed Pharmacia Mono Q HR 5/5column that was equilibrated in buffer C. The column was developed witha linear gradient of 0-2.0 M NaCl in buffer C at a flow rate of 0.5ml/min. Fractions that showed reductase activity were assayed forprotein content and stored at −80° C.

Rapid Enzyme Assays.

Activity for each of the three individual components was monitored inreactions using ¹⁴C-labeled dicamba in a reaction containing an excessof the remaining two components. For each reaction, a buffer solutioncomposed of 24 mM potassium phosphate (KP_(i)) buffer (pH 7.0), 0.48 mMNADH, 0.48 mM FeSO₄ and 9.6 mM MgCl₂ was added to 200 μl of proteinsample along with 30 μl of master mix (562.4 μl sterile water, 12.0 μl50 mM dicamba and 25.6 μl ¹⁴C-dicamba stock solution [1.28 μCi]) for atotal volume of 311 μl and a ¹⁴C-dicamba specific activity of 4.12μC_(i)/ml. After 60 min, each reaction was stopped by the addition of 50μl 5% H₂SO₄ (v/v) and 500 μl ether. Reaction tubes were vortexed andcentrifuged in a microfuge (Eppendorf, Model 5415C) at 14,000×g for 2minutes. For a quick visual appraisal of enzymatic activity, eachreaction tube was placed under a hand-held UV light (Fotodyne Inc.,Model 3-6000) to detect fluorescence of the reaction product, DCSA. Formore accurate, semi-quantitative measurements of enzymatic activities,reaction products were separated using thin layer chromatography asdescribed in Example 1.

Protein Concentration Determinations.

The fractions obtained at different stages of the purification protocolwere assayed for protein concentration using the Bradford assay(standard protocol; Bio-Rad; see Example 1).

Example 4 Identification and Sequencing of a Clone Coding forFerredoxin_(DIC)

The N-terminal sequence obtained from the purified ferredoxin protein(purification described in Example 3) was 29 amino acids in length(sequencing of proteins was performed by the Protein Core Facility atthe University of Nebraska-Lincoln using a standard Edman degradationprocedure; see Example 1). A comparison of this sequence to the Genbankdatabase showed that it was 35% identical in a 26 amino acid overlap toa terpredoxin from Pseudomonas sp., a bacterial [2Fe-2S] ferredoxin inthe adrenodoxin family. The sequence information was used to designthree degenerate oligonucleotide primers (two forward and one reverse).The sequence of the two 17 mer forward primers was based on theN-terminal amino acid sequence obtained from the purified ferredoxinprotein. The sequence of the 17 mer reverse primer was based on aconserved sequence of six amino acids near the C-terminal end of sixpreviously sequenced bacterial adrenodoxin-type ferredoxins. The primerswere used in a nested PCR reaction to amplify a 191 by product fromtotal P. maltophilia DNA. The product was cloned into the pGEM-T Easyvector (Promega, Madison, Wis.) and sequenced. DNA sequencing wasperformed by the DNA Sequencing Core Facility at the University ofNebraska-Lincoln using a standard dideoxy-mediated chain terminationprocedure. An analysis of the predicted amino acid sequence of thisclone confirmed that it matched the N-terminal and internal amino acidsequence previously obtained from the purified ferredoxin protein.Furthermore, the derived amino acid sequence had 48% identity over itsentire length with a [2Fe-2S] ferredoxin from Rhodobacter capsulatus.The cloned fragment was labeled with digoxigenin (DIG) (RocheDiagnostics) using a standard PCR protocol (DIG/Genius System User'sGuide) and hybridized by a Southern blot to total P. maltophilia DNAthat had been digested with a number of restriction enzymes. A map ofthe restriction sites surrounding the gene was constructed based on thesizes of the restriction fragments that hybridized to the probe. Thisinitial experiment showed that the gene was contained on a Xho I/Pst Ifragment that was approximately 1 kb in length. Subsequently, total P.maltophilia DNA was digested with Xho I and Pst I, and the restrictionfragments were resolved on a gel. Fragments between 0.5 and 1.5 kb inlength were excised from the gel, ligated into the vector pBluescript IIKS+ (Stratagene, Inc.) and transformed into DH5α cells (Gibco BRL,Inc.). Bacterial colonies containing the size-fractionated library werescreened with the DIG-labeled probe and a 900 by Xho I/Pst I fragmentwas identified. Sequence analysis showed that this clone contained afull-length 318 by ferredoxin gene [SEQ ID NO:4] that encoded an 11.4kDa protein composed of 105 amino acid residues [SEQ ID NO:5]. The aminoacid sequence predicted by the cloned gene matched the N-terminal andinternal amino acid sequence previously obtained from the purifiedferredoxin protein. Furthermore, the predicted amino acid sequence washomologous over its entire length to five other members of theadrenodoxin family of [2Fe-2S] bacterial ferredoxins, ranging from 42%identity with a ferredoxin from Rhodobacter capsulatus to 35% identitywith a ferredoxin from Pseudomonas (see FIG. 2). The other threeferredoxins were from Caulobacter crescentus, Rhodococcus erythropolis,and Pseudomonas putida. Proteins in this family bind a single 2Fe-2Siron-sulfur cluster and have three conserved motifs. Motif 1 includesthree conserved cysteines which are 2Fe-2S ligands. Motif 2 contains acluster of negatively charged residues. Motif 3 includes the fourthconserved cysteine of the 2Fe-2S cluster.

Example 5 Identification and Sequencing of Clones Coding Reductase_(DIC)

Two reductase genes were cloned by the same approach that was used inExample 4 to clone the ferredoxin gene. The N-terminal sequence obtainedfrom the purified reductase protein (purification described in Example3) was 25 amino acids in length. A comparison of this sequence to theGenbank database showed that it was 90% identical in a 20 amino acidoverlap to a cytochrome P450-type reductase component of dioxindioxygenase, a three component enzyme previously isolated fromSphingomonas sp. RW1. An internal sequence of 10 amino acid residues wasalso obtained from tryptic digests of the purified protein. The internalsequence had 87.5% identity with residues 62 through 69 of the samecytochrome P450-type reductase from Sphingomonas sp. RW1. This sequenceinformation was used to design three degenerate oligonucleotide primers(two forward and one reverse). The sequence of the two 17 mer forwardprimers was based on the N-terminal amino acid sequence and the sequenceof the 17 mer reverse primer was based on the internal amino acidsequence. The primers were used in a nested PCR reaction to amplify a180 by product from total P. maltophilia DNA. The product was clonedinto the pGEM-T Easy vector and sequenced. An analysis of the predictedamino acid sequence of this clone confirmed that it encoded a proteinthat matched the N-terminal and internal amino acid sequence obtainedfrom the purified reductase protein. Furthermore, the predicted sequencehad 80% identity over its entire length with the cytochrome P-450 typereductase component of the dioxin dioxygenase from Sphingomonas sp. RW1.

The cloned fragment was labeled with DIG and hybridized by a Southernblot to total P. maltophilia DNA that had been digested with a number ofrestriction enzymes. The Southern blot showed that the DIG-labeled proberecognized two distinct loci in the various restriction digests of P.maltophilia total DNA. This observation suggested that there are tworeductase genes located at different positions in the genome of P.maltophilia. It was possible that the two genes are identicalduplications that encode identical reductase proteins. Alternatively,one of the genes could encode a truncated protein with no activity or afull-length protein with low activity in our dicamba O-demethylaseassay. Because it was necessary to clone the gene that encodes a proteinwith optimal activity, the DIG-labeled probe was used to retrieve bothreductase genes.

When total P. maltophilia DNA was digested with Kpn I and EcoR I, theDIG-labeled probe hybridized to one restriction fragment that wasapproximately 4.0 kb in length and to another larger fragment with asize of approximately 20 kb. A map of a number of restriction sitessurrounding the gene located on the 4.0 kb Kpn I/EcoR I fragment wasconstructed based on the sizes of different restriction fragments thathybridized to the probe. The restriction map indicated that the entiregene should be located on this 4.0 kb fragment. Subsequently, total P.maltophilia DNA was digested with Kpn I and EcoR I, and the restrictionfragments were resolved on a gel. Fragments between 3.0 and 5.0 kb inlength were excised from the gel, ligated into the vector pBluescript IIKS+, and transformed into DH5α cells. Bacterial colonies containing thesize-fractionated library were screened with the DIG-labeled probe and a4.3 kb Kpn I/EcoR I fragment was identified. Sequence analysis showedthat this clone contained a full-length 1224 by reductase gene [SEQ IDNO:6] and encoded a 43.7 kDa protein consisting of 408 amino acids [SEQID NO:7]. The amino acid sequence predicted by the cloned gene matchedthe N-terminal and internal amino acid sequence previously obtained fromthe purified reductase protein. Furthermore, the predicted amino acidsequence was homologous over its entire length to at least four otherFAD-dependent pyridine nucleotide reductases, ranging from 69% identitywith a cytochrome P450-type reductase component of dioxin dioxygenasefrom Sphingomonas sp. RW1 to 36% identity with the terpredoxin reductasefrom a Pseudomonas species (see FIG. 3). The two other FAD-dependentpyridine nucleotide reductases were from R. erythropolis and P. putida.Proteins in this family of FAD-dependent pyridine nucleotide reductaseshave five conserved motifs. Motifs 1 and 3 contain three conservedglycine residues and correspond to the ADP binding site for FAD andNAD(P) respectively. Motif 5 corresponds to the site binding the FADflavin moiety.

To clone the second gene, total P. maltophilia DNA was digested with KpnI and EcoR I and the resulting restriction fragments were resolved on anagarose gel. Fragments with a size of approximately 20 kb were excisedfrom the gel, digested with a number of restriction enzymes, and thenhybridized by Southern blot to the DIG-labeled probe. A map of therestriction sites surrounding the second gene was constructed based onthe sizes of the restriction fragments that hybridized to the probe.These experiments showed that the full-length second reductase gene wascontained on an Apa I fragment that was approximately 3.0 kb in length.Subsequently, total P. maltophilia DNA was digested with Apa I and therestriction fragments were resolved on a gel. Fragments between 2.0 and4.0 kb in length were excised from the gel, ligated into the vectorpBluescript II KS+, and transformed into DH5α cells. Bacterial coloniescontaining the size-fractionated library were screened with theDIG-labeled probe and a 3.0 kb Apa I fragment was identified. Sequenceanalysis showed that the 3.0 kb clone contained an open reading frame of1227 by [SEQ ID NO:8] and encoded a 43.9 kDa protein consisting of 409amino acids [SEQ ID NO:9]. The amino acid sequence encoded by the secondreductase gene is almost identical (98.8% identity) to the sequence ofthe first gene.

Example 6 Transformation of Plants

In order to place each of the three genes that encode the components ofdicamba O-demethylase individually into cassettes suitable forexpression in plants, the following steps were taken. Oligonucleotideprimers were designed to generate a Nco I site at the 5′ end and an XbaI site at the 3′ end of each of the three genes by PCR amplification.The authenticity of the resulting PCR products was confirmed bysequencing, and each gene was then cloned individually into the pRTL2vector (provided by Dr. Tom Clemente of the Plant Transformation CoreResearch Facility, University of Nebraska, Lincoln, Neb.). This vectorcontains a 144 by translation enhancer sequence from tobacco etch virus(TEV) (Carrington and Freed, J. Virology, 64:1590-1597 (1990)) at the 5′end of the polylinker. The oxygenase, reductase, and ferredoxin genes,each with a 5′ translation enhancer, were then cloned individually asXho I/Xba I fragments into the plant expression vector pKLP36 (obtainedfrom Indu Maiti, University of Kentucky, Lexington, Ky.) (Maiti andShepard, Biochem. Biophys. Res. Commun., 244:440-444 (1998)). Thisbinary vector contains the peanut chlorotic virus full-length promoter(PC1SV FLt36) with a duplicated enhancer domain for constitutiveexpression in plants and the pea rbcS 3′ sequence for efficienttranscript termination (Maiti and Shepherd, Biochem. Biophys. Res.Commun., 244:440-444 (1998)). Constructs with all three genes on onebinary vector can be produced using combinations of the three genes in anumber of different orders and orientations.

The following methods were employed to move the oxygenase, ferredoxin,and reductase constructs individually into Agrobacterium tumefaciens andto transform each construct into Arabidopsis and tobacco. The threeconstructs were moved into the A. tumefaciens strain C58C1 by a modifiedtriparental mating procedure routinely used by the Plant TransformationCore Research Facility. This involved incubating Escherichia coli cellscarrying each construct with a mixture of A. tumefaciens cells and E.coli cells carrying the helper plasmid pRK2013 (for plasmidmobilization). A. tumefaciens cells containing each of the constructswere then used to transform tobacco and Arabidopsis with the assistanceof the Plant Transformation Core Research Facility. For tobacco, leafexplants were incubated with a suspension of A. tumefaciens cellscontaining each of the constructs, and shoots were regenerated on solidmedium containing kanamycin (Horsch et al., Science, 227:1229-1231(1985)). Ten shoots were selected from each of the three transformationexperiments, placed on rooting medium for a few weeks, and then moved topots in the greenhouse. For Arabidopsis, a pot of plants with flowerswas incubated with a suspension of A. tumefaciens cells containing eachof the constructs, and the plants were then allowed to set seed(Bechtold et al., C.R. Acad. Sci. Paris, Sciences de la vie/Lifesciences, 316:1194-1199 (1993); Clough and Bent, Plant J., 16(6):735-743(1998)). The seed was collected and germinated on medium with kanamycin.After the seedlings had developed an adequate root system, severalplants were selected from each transformation experiment and moved topots in a growth chamber.

For evaluation of the expression of each gene in the transformed plants,Western blots of leaf lysates from several transformed plants wereprepared and probed with polyclonal antibodies that detect the threecomponents of dicamba O-demethylase. To determine enzyme activities foreach enzyme in plant extracts, the approach was to combine leaf lysatesfrom transformed plants expressing the oxygenase, ferredoxin orreductase proteins with excess amounts of the other O-demethylasecomponents purified from P. maltophilia strain DI-6. These mixtures weretested for dicamba O-demethylase activity with both the standard¹⁴C-labeled dicamba assay (see Examples 1 and 3) and an HPLC assayemploying nonradioactive dicamba as substrate (dicamba and 3,6-DCSAelute at different points from the HPLC and can be quantitated).

To test expression of gene products in the chloroplast compartment,constructs were made with a transit peptide sequence at the 5′ end ofthe oxygenase, ferredoxin, and reductase genes. Such an approach wassuccessful for introducing tolerance to the sulfonylurea herbicides intotobacco plants (O'Keefe et al., Plant Physiol., 105:473-478 (1994)).

To test the possibility that the codon usage of the bacterial ferredoxingene was not fully optimal for efficient translation in a plant cell, asynthetic ferredoxin gene encoding the same amino acid sequence as theP. maltophilia strain DI-6 ferredoxin, but with optimized codon bias fordicot plants was synthesized by Entelechon, GmbH (Regensberg, Germany).It has been well documented that changes in codon usage are essentialfor optimal expression of the bacterial B.t. toxin genes in plant cells(see, e.g., Diehn et al., Genetic Engineering, 18:83-99 (1996)).

We claim:
 1. A method for producing a dicamba-degrading oxygenase thatcomprises the amino acid sequence of SEQ ID NO:3, or that comprises anamino acid sequence that differs from SEQ ID NO:3 by one substitution,an amino acid sequence that differs from SEQ ID NO:3 by one addition, oran amino acid sequence that differs from SEQ ID NO:3 by one substitutionand by one addition, said method comprising culturing a host celltransformed with a replication or expression vector comprising DNAsequence encoding a dicamba-degrading oxygenase that comprises the aminoacid sequence of SEQ ID NO:3, or that comprises an amino acid sequencethat differs from SEQ ID NO:3 by one substitution, an amino acidsequence that differs from SEQ ID NO:3 by one addition, or an amino acidsequence that differs from SEQ ID NO:3 by one substitution and by oneaddition, under conditions effective to express said polypeptide, andrecovering the polypeptide so expressed.
 2. A seed containing arecombinant dicamba-degrading oxygenase comprising the amino acidsequence of SEQ ID NO:3.
 3. A seed containing a recombinantdicamba-degrading oxygenase comprising an amino acid sequence thatdiffers from SEQ ID NO:3 by one substitution.
 4. A seed containing arecombinant dicamba-degrading oxygenase comprising an amino acidsequence that differs from SEQ ID NO:3 by one addition.
 5. A seedcontaining a recombinant dicamba-degrading oxygenase comprising an aminoacid sequence that differs from SEQ ID NO:3 by one substitution and byone addition.
 6. A seed of claim 2 wherein said oxygenase is produced byexpression of DNA comprising SEQ ID NO:
 2. 7. A seed of claim 3 whereinsaid oxygenase is produced by expression of a DNA encoding an oxygenasecomprising an amino acid sequence that differs from SEQ ID NO:3 by onesubstitution.
 8. A seed of claim 4 wherein said oxygenase is produced byexpression of a DNA encoding an oxygenase comprising an amino acidsequence that differs from SEQ ID NO:3 by one addition.
 9. A seed ofclaim 5 wherein said oxygenase is produced by expression of a DNAencoding an oxygenase comprising an amino acid sequence that differsfrom SEQ ID NO:3 by one substitution and by one addition.